Nucleic acid-containing lipid particles and related methods

ABSTRACT

Lipid particles containing a nucleic acid, devices and methods for making the lipid particles, and methods for using the lipid particles.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/049,872, filed Jul. 31, 2018, which is a continuation of U.S.application Ser. No. 15/687,218, filed Aug. 25, 2017 (now U.S. Pat. No.10,041,091), which is a continuation of U.S. application Ser. No.13/464,690, filed May 4, 2012 (now U.S. Pat. No. 9,758,795), which is acontinuation of International Application No. PCT/CA2010/001766, filedNov. 4, 2010, which claims the benefit of U.S. Provisional ApplicationNo. 61/280,510, filed Nov. 4, 2009, each of which is expresslyincorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 64113_Seq_Final.txt. The text file is 1.02 KB;was created on Aug. 24, 2017; and is being submitted via EFS-Web withthe filing of the specification.

BACKGROUND OF THE INVENTION

Lipid nanoparticles (LNP) are the most clinically advanced drug deliverysystems, with seven LNP-based drugs having received regulatory approval.These approved drugs contain small molecules such as anticancer drugsand exhibit improved efficacy and/or reduced toxicity compared to the“free” drug. LNP carrier technology has also been applied to delivery of“genetic” drugs such as plasmids for expression of therapeutic proteinsor small interfering RNA (siRNA) oligonucleotides (OGN) for silencinggenes contributing to disease progression. Devising methods forefficient in vivo delivery of siRNA OGN and other genetic drugs is themajor problem impeding the revolutionary potential of these agents astherapeutics.

Recent advances in LNP technology and the design of the cationic lipidsrequired for encapsulation and delivery of genetic drugs highlight thepotential of LNP systems to solve the in vivo delivery problem.LNP-siRNA systems have been shown to induce silencing of therapeuticallyrelevant target genes in animal models, including non-human primatesfollowing intravenous (i.v.) injection and are currently underevaluation in several clinical trials.

A variety of methods have been developed to formulate LNP systemscontaining genetic drugs. These methods include mixing preformed LNPwith OGN in the presence of ethanol or mixing lipid dissolved in ethanolwith an aqueous media containing OGN and result in LNP with diameters of100 nm or less and OGN encapsulation efficiencies of 65-95%. Both ofthese methods rely on the presence of cationic lipid to achieveencapsulation of OGN and poly(ethylene glycol) (PEG) lipids to inhibitaggregation and the formation of large structures. The properties of theLNP systems produced, including size and OGN encapsulation efficiency,are sensitive to a variety of formulation parameters such as ionicstrength, lipid and ethanol concentration, pH, OGN concentration andmixing rates. In general, parameters such as the relative lipid and OGNconcentrations at the time of mixing, as well as the mixing rates aredifficult to control using current formulation procedures, resulting invariability in the characteristics of LNP produced, both within andbetween preparations.

Microfluidic devices provide an ability to controllably and rapidly mixfluids at the nanoliter scale with precise control over temperature,residence times, and solute concentrations. Controlled and rapidmicrofluidic mixing has been previously applied in the synthesis ofinorganic nanoparticles and microparticles, and can outperformmacroscale systems in large scale production of nanoparticles.Microfluidic two-phase droplet techniques have been applied to producemonodisperse polymeric microparticles for drug delivery or to producelarge vesicles for the encapsulation of cells, proteins, or otherbiomolecules. The use of hydrodynamic flow focusing, a commonmicrofluidic technique to provide rapid mixing of reagents, to createmonodisperse liposomes of controlled size has been demonstrated. Thistechnique has also proven useful in the production of polymericnanoparticles where smaller, more monodisperse particles were obtained,with higher encapsulation of small molecules as compared to bulkproduction methods.

Despite advances in the development of methods for LNP systemscontaining genetic drugs, a need exists for devices and methods forpreparing lipid nanoparticles containing therapeutic materials, as wellas improved lipid nanoparticles containing therapeutic materials. Thepresent invention seeks to fulfill this need and provides furtherrelated advantages.

SUMMARY OF THE INVENTION

In one aspect, the invention provides lipid particles comprising nucleicacids.

In one embodiment, the lipid particle comprises (a) one or more cationiclipids, (b) one or more second lipids, and (c) one or more nucleicacids, wherein the lipid particle comprises a substantially solid core,as defined herein.

In one embodiment, the cationic lipid is DLin-KC2-DMA. In certainembodiments, the particle comprises from about 30 to about 95 molepercent cationic lipid.

In one embodiment, the second lipid is PEG-c-DMA. In one embodiment, thesecond lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Incertain embodiments, the particle comprises from about 1 to about 10mole percent second lipid.

The nucleic acid can be a DNA, a RNA, a locked nucleic acid, a nucleicacid analog, or a plasmid capable of expressing a DNA or an RNA.

In another embodiment, the lipid particle comprises (a) one or morecationic lipids, (b) one or more neutral lipids, (c) one or morePEG-lipids, (d) one or more sterols; and (e) one or more nucleic acids,wherein the lipid particle comprises a substantially solid core, asdefined herein. In one embodiment, the cationic lipid is DLin-KC2-DMA.In one embodiment, the PEG-lipid is PEG-c-DMA. In one embodiment, theneutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Inone embodiment, the sterol is cholesterol. In one embodiment, nucleicacid is an siRNA.

In a further embodiment, the lipid particle consists of one or morecationic lipids, and one or more nucleic acids. In one embodiment, thelipid particle comprises a substantially solid core, as defined herein.In one embodiment, the cationic lipid is DLin-KC2-DMA. In oneembodiment, the nucleic acid is an siRNA.

In other aspects, the invention provides methods for using the lipidparticles.

In one embodiment, the invention provides a method for administering anucleic acid to a subject, comprising administering a lipid particle ofthe invention to a subject in need thereof.

In one embodiment, the invention provides a method for introducing anucleic acid into a cell, comprising contacting a cell with the lipidparticle of the invention.

In one embodiment, the invention provides a method for modulating theexpression of a target polynucleotide or polypeptide, comprisingcontacting a cell with the lipid particle of the invention, wherein thenucleic acid capable of modulating the expression of a targetpolynucleotide or polypeptide.

In one embodiment, the invention provides a method of treating a diseaseor disorder characterized by overexpression of a polypeptide in asubject, comprising administering to the subject the lipid particle ofthe invention, wherein the nucleic acid capable of silencing ordecreasing the expression of the polypeptide.

In other aspect, the invention provides a method for making lipidparticles.

In one embodiment, the invention provides a method for making lipidparticles containing a nucleic acid, comprising:

(a) introducing a first stream comprising a nucleic acid in a firstsolvent into a microfluidic device; wherein the device has a firstregion adapted for flowing one or more streams introduced into thedevice and a second region for mixing the contents of the one or morestreams with a microfluidic mixer;

(b) introducing a second stream comprising lipid particle-formingmaterials in a second solvent into the device to provide first andsecond streams flowing under laminar flow conditions, wherein the devicehas a first region adapted for flowing one or more streams introducedinto the microchannel and a second region for mixing the contents of theone or more streams, wherein the lipid particle-forming materialscomprise a cationic lipid, and wherein the first and second solvents arenot the same;

(c) flowing the one or more first streams and the one or more secondstreams from the first region of the device into the second region ofthe device; and

(d) mixing of the contents of the one or more first streams and the oneor more second streams flowing under laminar flow conditions in thesecond region of the device to provide a third stream comprising lipidnanoparticles with encapsulated nucleic acid.

In another embodiment, the invention provides a method for making lipidparticles containing a nucleic acid, comprising:

(a) introducing a first stream comprising a nucleic acid in a firstsolvent into a channel; wherein the device has a first region adaptedfor flowing one or more streams introduced into the channel and a secondregion for mixing the contents of the one or more streams;

(b) introducing a second stream comprising lipid particle-formingmaterials in a second solvent; wherein the channel has a first regionadapted for flowing one or more streams introduced into the channel anda second region for mixing the contents of the one or more streams;

(c) flowing the one or more first streams and the one or more secondstreams from the first region of the channel into the second region ofthe channel, while maintaining a physical separation of the two streams,wherein the one or more first streams and the one or more second streamsdo not mix until arriving at the second region of the channel; and

(d) mixing of the contents of the one or more first streams and the oneor more second streams flowing under laminar flow conditions in thesecond region of the microchannel to provide a third stream comprisinglipid nanoparticles with encapsulated nucleic acids.

In certain embodiments of the above methods, mixing the contents of theone or more first streams and the one or more second streams comprisesvarying the concentration or relative mixing rates of the one or morefirst streams and the one or more second streams.

In certain embodiments of the above methods, the methods furthercomprise diluting the third stream with an aqueous buffer. In certainembodiments, diluting the third stream comprises flowing the thirdstream and an aqueous buffer into a second mixing structure.

In certain embodiments of the above methods, the methods furthercomprise dialyzing the aqueous buffer comprising lipid particles withencapsulated nucleic acids to reduce the amount of the second solvent.

In certain embodiments of the above methods, the first solvent is anaqueous buffer. In certain embodiments of the above methods, the secondsolvent is an aqueous alcohol.

In certain embodiments of the above methods, mixing the contents of thefirst and second streams comprises chaotic advection. In certainembodiments of the above methods, mixing the contents of the first andsecond streams comprises mixing with a micromixer.

In certain embodiments of the above methods, the nucleic acidencapsulation efficiency is from about 90 to about 100%.

In certain embodiments of the above methods, mixing of the one or morefirst streams and the one or more second streams is prevented in thefirst region by a barrier. In certain embodiments, the barrier is achannel wall, sheath fluid, or concentric tubing.

In another aspect of the invention, devices for making lipid particlesare provided. In one embodiment, the invention provides a device forproducing a lipid particle encapsulating a nucleic acid, comprising:

(a) a first inlet for receiving a first solution comprising a nucleicacid in a first solvent;

(b) a first inlet microchannel in fluid communication with the firstinlet to provide a first stream comprising the nucleic acid in the firstsolvent;

(c) a second inlet for receiving a second solution comprising lipidparticle-forming materials in a second solvent;

(d) a second inlet microchannel in fluid communication with the secondinlet to provide a second stream comprising the lipid particle-formingmaterials in the second solvent; and

(e) a third microchannel for receiving the first and second streams,wherein the third microchannel has a first region adapted for flowingthe first and second streams introduced into the microchannel underlaminar flow conditions and a second region adapted for mixing thecontents of the first and second streams to provide a third streamcomprising lipid particles with encapsulated nucleic acid.

In one embodiment, the device further comprises means for diluting thethird stream to provide a diluted stream comprising stabilized lipidparticles with encapsulated nucleic acid. In certain embodiments, themeans for diluting the third stream comprises a micromixer.

In one embodiment, the microchannel has a hydrodynamic diameter fromabout 20 to about 300 um.

In one embodiment, the second region of the microchannel comprisesbas-relief structures. In one embodiment, the second region of themicrochannel has a principal flow direction and one or more surfaceshaving at least one groove or protrusion defined therein, the groove orprotrusion having an orientation that forms an angle with the principaldirection. In one embodiment, the second region comprises a micromixer.

In certain embodiments, the device further comprises means for varyingthe flow rates of the first and second streams.

In certain embodiments, the device further comprises a barrier effectiveto physically separate the one or more first streams from the one ormore second streams in the first region.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a schematic illustration of a representative fluidic device ofthe invention.

FIG. 2 is a schematic illustration of a representative fluidic device ofthe invention that is an elaboration of the device illustrated in FIG.1.

FIG. 3 is a schematic illustration of a representative fluidic device ofthe invention that is an elaboration of the device illustrated in FIG.2.

FIG. 4 is a schematic illustration of a representative fluidic deviceand method of the invention.

FIG. 5 is a schematic illustration of a representative array of theinvention comprising ten of the fluidic devices illustrated in FIG. 4.

FIG. 6 is a schematic illustration of a representative fluidic device ofthe invention.

FIG. 7 is a schematic illustration of a representative array of theinvention comprising ten of the representative fluidic devicesillustrated in FIG. 6.

FIG. 8 is a schematic illustration of a representative fluidic device ofthe invention having three inlets and a single outlet (device 800includes mixing channel 810).

FIG. 9 is a schematic illustration of a representative fluidic device ofthe invention having two inlets and a single outlet (device 900 includesmixing channel 910).

FIG. 10 is a schematic illustration of a representative fluidic deviceof the invention having a multiplicity (n) of serial inlets and a singleoutlet (device 1000 includes mixing channels 1010 a, 1010 b, 1010 c, and1010 d).

FIG. 11 is a schematic illustration of a representative fluidic deviceof the invention having three inlets and a single outlet (device 1100includes mixing channels 1110 a, 1110 b, and 1110 c).

FIG. 12 is a schematic illustration of a representative fluidic deviceof the invention having seven inlets and a single outlet (device 1200includes mixing channels 1210 a, 1210 b, 1210 c, and 1210 d).

FIG. 13 is a schematic illustration of a representative fluidic deviceof the invention having a multilaminate mixer (device 1300 includesmixing channel 1310).

FIG. 14 is a close-up view of the multilaminate mixer illustrated inFIG. 14.

FIG. 15A is a schematic illustration of a representative microfluidic(MF) method of the invention for making lipid nanoparticles (LNP):Lipid-ethanol and siRNA-aqueous solutions are pumped into inlets of amicrofluidic mixing device; herringbone features in the device inducechaotic advection of the laminar stream and cause the lipid species torapidly mix with the aqueous stream and form lipid nanoparticles. Themixing channel is 200 μm wide and 79 μm high. The herringbone structuresare 31 μm high and 50 μm thick.

FIG. 15B is a schematic illustration of a preformed vesicle (PFV) methodfor making lipid nanoparticles (LNP): (a) a lipid-ethanol solution isadded to an aqueous solution, pH 4.0, resulting in the formation ofvesicle type particles; (b) extrusion through 80 nm polycarbonatemembrane (Nuclepore) at room temperature using a Lipex Extruder providesa more uniform particle distribution; and (c) addition of siRNA solutionwhile vortexing and incubation at 35° C. for 30 minutes promotesencapsulation of siRNA.

FIGS. 16A-16C illustrate the influence of flow rate in microfluidicdevice on mixing and LNP particle size. Two 10 μM fluorescein(fluorescent at pH 8.8, non-fluorescent at pH 5.15) solutions mix toproduce completely fluorescent solution.

FIG. 16A compares the extent of mixing (%) as determined by meanfluorescent intensity along channel width as a function of with mixingtime (msec) calculated from average fluid velocity and travel length(0.2, 0.8, 1.4, and 2 mL/min). FIGS. 16B and 16C compare mean particlediameter for LNP composed of DLin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA atmole ratios of 40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt, with10 mM lipid-ethanol phase mixed with 25 mM acetate buffer, pH 4,containing siRNA. FIG. 16B compares mean particle diameter (nm) for LNPas a function of flow rate (mL/min). FIG. 16C compares mean particlediameter (nm) for LNP as a function of ethanol/aqueous flow rate ratio.Error bars represent standard deviation of the mean particle diameter asmeasured by dynamic light scattering.

FIG. 17 illustrates the influence of lipid concentration on LNP particlesize by comparing mean particle diameter (nm) as a function of lipidconcentration in ethanol (mM). Increasing the lipid concentrationresults in an increase in mean particle diameter. The total lipidcontent in the ethanol phase being mixing in the microfluidic chip wasvaried from 10 mM to 50 mM. LNP composed ofDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Total flow rateinside microfluidic mixer was maintained at 2 ml/min Error barsrepresent standard deviation of the mean particle diameter as measuredby dynamic light scattering.

FIGS. 18A and 18B illustrate the influence of PEG-lipid and cationiclipid on LNP systems. FIG. 18A compares mean particle diameter (nm) as afunction of PEG-c-DMA content (mol % in LNP) for LNP prepared by the PFVand MF methods. The PEG-lipid was varied from 1 mol % to 10 mol % in theLNP composition. Modification of PEG-lipid content was compensated byadjustment of cholesterol content. LNP were composed ofDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of 40:11.5:47.5:1(−x):1 (+x), (where x=1 to 9), siRNA-total lipid ratio 0.06 wt/wt. FIG.18B compares mean particle diameter (nm) as a function of DLin-KC2-DMAcontent (mol %) for LNP prepared by the PFV and MF methods. The cationiclipid was varied from 40 mol % to 70 mol %. PEG-c-DMA was kept constantat 1 mol % and a 0.25 molar ratio was maintained with DSPC-cholesterol.Total flow rate inside microfluidic mixer was maintained at 2 ml/min 10mM lipid-ethanol phase mixed with 25 mM acetate buffer, pH 4, containingsiRNA. Error bars represent standard deviation of the mean particlediameter as measured by dynamic light scattering.

FIG. 19 illustrates the influence of siRNA/Lipid ratio on particle sizeand encapsulation by comparing mean particle diameter (nm) andencapsulation (%) as a function of siRNA/lipid ratio (wt/wt) (alsoexpressed as nucleotide/phosphate (N/P).

Encapsulation determined by separation of LNP suspension from free siRNAusing an anionic exchange spin column LNP were composed ofDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Total flow rateinside microfluidic mixer was maintained at 2 ml/min 10 mM lipid-ethanolphase mixed with 25 mM acetate buffer, pH 4, containing siRNA. Errorbars represent standard deviation of the mean particle diameter asmeasured by dynamic light scattering.

FIGS. 20A and 20B illustrate the morphology of PEG-lipid and cationiclipid LNP systems prepared by the microfluidic mixer usingCryo-Transmission Electron Microscopy (TEM). LNP were imaged at 29Kmagnification by Cryo-TEM.

FIG. 20A is an image of empty LNP composed ofDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of40:11.5:47.5:1. FIG. 20B is an image of siRNA loaded LNP composed ofDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Formulation wasperformed using the microfluidic mixer at 20 mM lipid in the ethanolphase. Loaded LNP-siRNA and empty particles containing 1 mol %PEG-c-DOMG exhibited identical morphology and are very homogeneous instructure. Scale bar represents 100 nm.

FIG. 21 illustrates in vivo silencing activity of microfluidic producedLNP in Factor VII Mouse Model by comparing relative FVII Protein Level(%) as a function of siRNA dosage (mg/kg) varying DLin-KC2-DMA contentin the LNP from 40 mol % to 60 mol %. Formulation of LNP containing 1mol % PEG-c-DOMG and 60 mol % DLin-KC2-DMA provide FVII silencingsimilar to that previously reported using alternative approaches. Genesilencing progressively improves for LNP containing DLin-KC2-DMA overthe range from 40 mol % to 60 mol %. Systemic injection of LNP-siRNA tomice was performed by tail vein injection (n=3 per dose level). Bloodcollection was performed after 24 hrs post-injection and factor VIIlevels were determined by colorimetric assay. LNP DSPC-to-Cholesterolratio was kept at 0.2 wt/wt and contained 1 mol % PEG-c-DOMG. LNPsiRNA-to-lipid ratio was 0.06 wt/wt.

FIGS. 22A-22C illustrate cryo electron microscopy of lipid nanoparticlesprepared by the microfluidics method. Empty lipid nanoparticles preparedby microfluidics (40% DLinKC2-DMA, 11.5% DSPC, 47.5% cholesterol, 1%PEG-c-DMA) showed an electron dense interior indicating solid corestructure (FIG. 22A). Samples composed with POPC showed a less denseinterior correlating with aqueous core vesicles (FIG. 22B). Systemscontaining POPC/triolein which have a hydrophobic core of trioleinsurrounded by a monolayer of POPC showed an electron dense interiorsimilar to sample A (FIG. 22C).

FIG. 23 illustrates limit size LNP prepared with DLinKC2-DMA/PEG-lipidsystem (90/10, mol/mol) using microfluidic mixing by comparing meanparticle diameter (nm) as a function of ethanol/aqueous flow rate ratiofor LNP were produced in the presence of siRNA (N/P=1) and without siRNApresent (No siRNA). Formulation was performed using a 10 mMlipid-ethanol phase mixed with 25 mM acetate buffer, pH 4. The particlesize was determined by dynamic light scattering and number-weighted meandiameters are reported.

FIGS. 24A-24C illustrate ³¹P NMR of siRNA encapsulated in 50%DLinKC2-DMA, 45% cholesterol, and 5% PEG-c-DMA using microfluidicmixing. DSPC was omitted to avoid conflicting phosphorus signal arisingfrom the phospholipid. ³¹P signal from the siRNA cannot be detected forintact LNP (FIG. 24A) or after the addition of 150 mM ammonium acetate(FIG. 24B). Signal can only be detected after the addition of 1% SDS tosolubilize the particle (FIG. 24C).

FIG. 25 is an electrophoretic gel illustrates the results of an RNaseprotection assay. siRNA was encapsulated using either the microfluidicmethod (MF) or the PFV approach, or left unencapsulated. Triton X-100was added to completely solubilize and lyse the lipid particles. Gelelectrophoresis was performed on 20% native polyacrylamide gel and siRNAvisualized by staining with CYBR-Safe.

FIG. 26 illustrates the results of a lipid mixing fusion assayrepresented as percent lipid mixing as a function of time (seconds). Toassess the amount of exposed cationic lipid present in the outermostlayer of the LNP, three LNP systems were prepared: in the absence ofsiRNA (No siRNA), at N/P=4, and N/P=1. Lipid assay was performed at pH5.5 to ensure nearly complete ionization of the cationic lipid, and thereaction was initiated by injecting the LNP into the cuvette containinghighly anionic DOPS/NBD-PE/Rh-PE (98:1:1 molar ratio) vesicles.

FIG. 27 is a schematic representation of the solid core LNP siRNA systemformed by microfluidic mixing in accordance with the method of theinvention.

FIGS. 28A and 28B illustrate mean particle diameter (nm) and zetapotential (mV), respectively, as a function of sequential lipidnanoparticle composition prepared using the microfluidic mixer.

FIG. 29 is a schematic representation of a representative device andmethod of the invention for the sequential assembly of lipidnanoparticles.

FIG. 30 is a schematic representation of a representative device andmethod of the invention.

FIG. 31 is a schematic representation of a representative device andmethod of the invention.

FIG. 32 is a schematic representation of a representative device andmethod of the invention.

FIG. 33 is a schematic representation of a representative device andmethod of the invention.

FIGS. 34A and 34B compare cryo-transmission electron microscopy imagesof two LNPs. LNP produced by the microfluidic method of the inventionresult in small spherical particles. As shown in FIG. 34A LNP composedof pure DOPC produced by the microfluidics procedure are very small“limit size” vesicles in the range of 30-50 nm diameter. The vesicleshave an interior with lower electron density consistent with a bilayershell surrounding an inner aqueous core. Interestingly, LNP consistingof DOPE/DOPC/PEG-lipid produced by the microfluidics procedure of theinvention (FIG. 34B) exhibit somewhat larger sizes where the majority ofthe particles have an electron dense inner core. This could beconsistent with the formation of solid core particles where lipids withthe lowest solubility in water (e.g., DOPE) condense out to formnucleation points that are subsequently coated by more polar lipids suchas DOPC and PEG-lipid. Such a process may be occurring for the cationiclipids and cationic lipid-polynucleic acid systems in the lipid mixturesemployed for polynucleic acid entrapment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides lipid particles containing a therapeuticagent, methods and devices for making the lipid particles containing atherapeutic agent, and methods for delivering a therapeutic agent usingthe lipid particles.

Lipid Particles

In one aspect, the invention provides lipid particles containing atherapeutic agent. The lipid particles include one or more cationiclipids, one or more second lipids, and one or more nucleic acids.

Cationic lipid. The lipid particles include a cationic lipid. As usedherein, the term “cationic lipid” refers to a lipid that is cationic orbecomes cationic (protonated) as the pH is lowered below the pK of theionizable group of the lipid, but is progressively more neutral athigher pH values. At pH values below the pK, the lipid is then able toassociate with negatively charged nucleic acids (e.g.,oligonucleotides). As used herein, the term “cationic lipid” includeszwitterionic lipids that assume a positive charge on pH decrease.

The term “cationic lipid” refers to any of a number of lipid specieswhich carry a net positive charge at a selective pH, such asphysiological pH. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE). Additionally, a number of commercial preparations ofcationic lipids are available which can be used in the presentinvention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, GrandIsland, NY); LIPOFECTAMINE® (commercially available cationic liposomescomprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

The lipid particle-forming materials include an ionizable lipid. As usedherein, the term “ionizable lipid” refers to a lipid that becomescationic (protonated) as the pH is lowered below the pK of the ionizablegroup of the lipid, but is progressively more neutral at higher pHvalues. At pH values below the pK, the lipid is then able to associatewith negatively charged polynucleic acids (e.g., oligonucleotides). Inone embodiment, the ionizable lipid is an amino lipid.

In one embodiment, the cationic lipid is an amino lipid. Suitable aminolipids useful in the invention include those described in WO2009/096558, incorporated herein by reference in its entirety.Representative amino lipids include1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3 -morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

Suitable amino lipids include those having the formula:

wherein R₁ and R₂ are either the same or different and independentlyoptionally substituted C₁₀-C₂₄ alkyl, optionally substituted C₁₀-C₂₄alkenyl, optionally substituted C₁₀-C₂₄ alkynyl, or optionallysubstituted C₁₀-C₂₄ acyl;

R₃ and R₄ are either the same or different and independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, oroptionally substituted C₂-C₆ alkynyl or R₃ and R₄ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R₅ is either absent or present and when present is hydrogen or C₁-C₆alkyl;

m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0;

q is0, 1,2, 3,or4; and

Y and Z are either the same or different and independently O, S, or NH.

In one embodiment, R₁ and R₂ are each linoleyl, and the amino lipid is adilinoleyl amino lipid. In one embodiment, the amino lipid is adilinoleyl amino lipid.

A representative useful dilinoleyl amino lipid has the formula:

wherein n is 0, 1, 2, 3, or 4.

In one embodiment, the cationic lipid is a DLin-K-DMA. In oneembodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above,wherein n is 2).

Other suitable cationic lipids include cationic lipids, which carry anet positive charge at about physiological pH, in addition to thosespecifically described above, N,N-dioleyl-N,N-dimethylammonium chloride(DODAC); N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride(DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);1,2-dioleyloxy-3 -trimethylaminopropane chloride salt (DOTAP.Cl);3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol),N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL).

The cationic lipid is present in the lipid particle in an amount fromabout 30 to about 95 mole percent. In one embodiment, the cationic lipidis present in the lipid particle in an amount from about 30 to about 70mole percent. In one embodiment, the cationic lipid is present in thelipid particle in an amount from about 40 to about 60 mole percent.

In one embodiment, the lipid particle includes (“consists of”) only ofone or more cationic lipids and one or more nucleic acids. Thepreparation and characterization of a lipid particle of the inventionconsisting of a cationic lipid and a nucleic acid is described inExample 5.

Other suitable ionizable lipids include DODAC, DOTMA, DDAB, DOTAP,DOTAP.Cl, DC-Chol, DOSPA, DOGS, DOPE, DODAP, DODMA, DODMA, and DMRIE,among others. See, for example, lipids described in WO 2009/096558,expressly incorporated herein by reference in its entirety.

In addition to the ionizable lipid, the second stream includes one ormore other lipid particle-forming materials. Representative lipidparticle-forming materials include polyethylene glycol-lipids, neutrallipids, and sterols.

Second lipids. In certain embodiments, the lipid particles include oneor more second lipids. Suitable second lipids stabilize the formation ofparticles during their formation.

The term “lipid” refers to a group of organic compounds that are estersof fatty acids and are characterized by being insoluble in water butsoluble in many organic solvents. Lipids are usually divided in at leastthree classes: (1) “simple lipids” which include fats and oils as wellas waxes; (2) “compound lipids” which include phospholipids andglycolipids; and (3) “derived lipids” such as steroids.

Suitable stabilizing lipids include neutral lipids and anionic lipids.Neutral Lipid. The term “neutral lipid” refers to any one of a number oflipid species that exist in either an uncharged or neutral zwitterionicform at physiological pH. Representative neutral lipids includediacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

Exemplary lipids include, for example, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).

In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

Anionic Lipid. The term “anionic lipid” refers to any lipid that isnegatively charged at physiological pH. These lipids includephosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dodecanoylphosphatidylethanol-amines,N-succinylphosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

Other suitable lipids include glycolipids (e.g., monosialogangliosideGM₁). Other suitable second lipids include sterols, such as cholesterol.

Polyethylene glycol-lipids. In certain embodiments, the second lipid isa polyethylene glycol-lipid. Suitable polyethylene glycol-lipids includePEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid,PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modifieddialkylamines, PEG-modified diacylglycerols, PEG-modifieddialkylglycerols. Representative polyethylene glycol-lipids includePEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, thepolyethylene glycol-lipid is N-[(methoxy poly(ethyleneglycol)₂₀₀₀)carbamy]1-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Inone embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).

Representative polyethylene glycol-lipids include PEG-modifiedphosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modifiedceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols,PEG-modified dialkylglycerols, PEG-C-DOMG, PEG-DMA, and PEG-s-DMG. See,for example, PEG-lipids described in WO 2009/096558, expresslyincorporated herein by reference in its entirety.

Advantageously, the lipid particles include from about 1 to about 5 molepercent PEG-lipid. In one embodiment, the lipid particles include about1 mole percent PEG-lipid.

In certain embodiments, the second lipid is present in the lipidparticle in an amount from about 1 to about 10 mole percent. In oneembodiment, the second lipid is present in the lipid particle in anamount from about 1 to about 5 mole percent. In one embodiment, thesecond lipid is present in the lipid particle in about 1 mole percent.

Nucleic Acids. The lipid particles of the present invention are usefulfor the systemic or local delivery of nucleic acids. As describedherein, the nucleic acid is incorporated into the lipid particle duringits formation.

As used herein, the term “nucleic acid” is meant to include anyoligonucleotide or polynucleotide. Fragments containing up to 50nucleotides are generally termed oligonucleotides, and longer fragmentsare called polynucleotides. In particular embodiments, oligonucleotidesof the present invention are 20-50 nucleotides in length. In the contextof this invention, the terms “polynucleotide” and “oligonucleotide”refer to a polymer or oligomer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages. The terms “polynucleotide” and “oligonucleotide”also includes polymers or oligomers comprising non-naturally occurringmonomers, or portions thereof, which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases. Oligonucleotides areclassified as deoxyribooligonucleotides or ribooligonucleotides. Adeoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribosejoined covalently to phosphate at the 5′ and 3′ carbons of this sugar toform an alternating, unbranched polymer. A ribooligonucleotide consistsof a similar repeating structure where the 5-carbon sugar is ribose. Thenucleic acid that is present in a lipid particle according to thisinvention includes any form of nucleic acid that is known. The nucleicacids used herein can be single-stranded DNA or RNA, or double-strandedDNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA includestructural genes, genes including control and termination regions, andself-replicating systems such as viral or plasmid DNA. Examples ofdouble-stranded RNA include siRNA and other RNA interference reagents.Single-stranded nucleic acids include antisense oligonucleotides,ribozymes, microRNA, and triplex-forming oligonucleotides. In oneembodiment, the polynucleic acid is an antisense oligonucleotide. Incertain embodiments, the nucleic acid is an antisense nucleic acid, aribozyme, tRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, pre-condensed DNA, oran aptamer.

The method of the invention provides lipid particles containing atherapeutic agent, such as a genetic material (e.g., a polynucleicacid). As used herein, the term “polynucleic acid” refers to a DNA, aRNA, a locked nucleic acid (LNA), or other nucleic acid analog known inthe art, or a plasmid capable of expressing a DNA or a RNA. In oneembodiment, the polynucleic acid is an oligonucleotide. The polynucleicacid may be a ssDNA or a dsDNA, an siRNA or a microRNA. In oneembodiment, the polynucleic acid is an antisense oligonucleotide.

The term “nucleic acids” also refers to ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backboneoligonucleotides, other nucleotides, nucleotide analogs, andcombinations thereof, and can be single stranded, double stranded, orcontain portions of both double stranded and single stranded sequence,as appropriate.

The term “nucleotide,” as used herein, generically encompasses thefollowing terms, which are defined below: nucleotide base, nucleoside,nucleotide analog, and universal nucleotide.

The term “nucleotide base,” as used herein, refers to a substituted orunsubstituted parent aromatic ring or rings. In some embodiments, thearomatic ring or rings contain at least one nitrogen atom. In someembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, purines such as 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N6-2-isopentenyladenine(6iA), N6-2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine,guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine(7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and06-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and4-methylindole; pyrroles such as nitropyrrole; nebularine; base (Y); Insome embodiments, nucleotide bases are universal nucleotide bases.Additional exemplary nucleotide bases can be found in Fasman, 1989,Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394,CRC Press, Boca Raton, Fla., and the references cited therein. Furtherexamples of universal bases can be found, for example, in Loakes, N.A.R.2001, 29:2437-2447 and Seela N.A.R. 2000, 28:3224-3232.

The term “nucleoside,” as used herein, refers to a compound having anucleotide base covalently linked to the C-1′ carbon of a pentose sugar.In some embodiments, the linkage is via a heteroaromatic ring nitrogen.Typical pentose sugars include, but are not limited to, those pentosesin which one or more of the carbon atoms are each independentlysubstituted with one or more of the same or different —R, —OR, —NRR orhalogen groups, where each R is independently hydrogen, (C1-C6) alkyl or(C5-C14) aryl. The pentose sugar may be saturated or unsaturated.Exemplary pentose sugars and analogs thereof include, but are notlimited to, ribose, 2′-deoxyribose, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-dideoxyribose, 2′,3′-didehydroribose,2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose,2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose, 2′-deoxy-3(C1-C6)alkylribose, 2′-deoxy-3′-(C1-C6)alkoxyribose and2′-deoxy-3′-(C5-C14)aryloxyribose. Also see, e.g., 2′-0-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides (Asseline (1991)Nucl. Acids Res. 19:4067-74), 2′-4′- and 3′-4′-linked and other “locked”or “LNA,” bicyclic sugar modifications (WO 98/22489; WO 98/39352; WO99/14226). “LNA” or “locked nucleic acid” is a DNA analogue that isconformationally locked such that the ribose ring is constrained by amethylene linkage between the 2′-oxygen and the 3′- or 4′-carbon. Theconformation restriction imposed by the linkage often increases bindingaffinity for complementary sequences and increases the thermal stabilityof such duplexes.

Sugars include modifications at the 2′- or 3′-position such as methoxy,ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy,phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo. Nucleosidesand nucleotides include the natural D configurational isomer (D-form),as well as the L configurational isomer (L-form) (Beigelman, U.S. Pat.No. 6,251,666; Chu, U.S. Pat. No. 5,753,789; Shudo, EP0540742; Garbesi(1993) Nucl. Acids Res. 21:4159-65; Fujimori (1990) J. Amer. Chem. Soc.112:7435; Urata, (1993) Nucleic Acids Symposium Ser. No. 29:69-70). Whenthe nucleobase is purine, e.g., A or G, the ribose sugar is attached tothe N9-position of the nucleobase. When the nucleobase is pyrimidine,e.g., C, T or U, the pentose sugar is attached to the N1-position of thenucleobase (Kornberg and Baker, (1992) DNA Replication, 2^(nd) Ed.,Freeman, San Francisco, Calif.).

One or more of the pentose carbons of a nucleoside may be substitutedwith a phosphate ester. In some embodiments, the phosphate ester isattached to the 3′- or 5′-carbon of the pentose. In some embodiments,the nucleosides are those in which the nucleotide base is a purine, a7-deazapurine, a pyrimidine, a universal nucleotide base, a specificnucleotide base, or an analog thereof.

The term “nucleotide analog,” as used herein, refers to embodiments inwhich the pentose sugar and/or the nucleotide base and/or one or more ofthe phosphate esters of a nucleoside may be replaced with its respectiveanalog. In some embodiments, exemplary pentose sugar analogs are thosedescribed above. In some embodiments, the nucleotide analogs have anucleotide base analog as described above. In some embodiments,exemplary phosphate ester analogs include, but are not limited to,alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, and mayinclude associated counterions. Other nucleic acid analogs and basesinclude for example intercalating nucleic acids (INAs, as described inChristensen and Pedersen, 2002), and AEGIS bases (Eragen, U.S. Pat. No.5,432,272). Additional descriptions of various nucleic acid analogs canalso be found for example in (Beaucage et al., Tetrahedron 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970);Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl.Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984),Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al.,Chemica Scripta 26:141 (1986)), phosphorothioate (Mag et al., NucleicAcids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048. Other nucleicanalogs comprise phosphorodithioates (Briu et al., J. Am. Chem. Soc.111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), those with positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023;5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi etal., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J.Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &Nucleotide 13:1597 (194): Chapters 2 and 3, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research,” Ed. Y. S. Sanghuiand P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research,” Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)pp. 169-176). Several nucleic acid analogs are also described in Rawls,C & E News Jun. 2, 1997, page 35.

The term “universal nucleotide base” or “universal base,” as usedherein, refers to an aromatic ring moiety, which may or may not containnitrogen atoms. In some embodiments, a universal base may be covalentlyattached to the C-1′ carbon of a pentose sugar to make a universalnucleotide. In some embodiments, a universal nucleotide base does nothydrogen bond specifically with another nucleotide base. In someembodiments, a universal nucleotide base hydrogen bonds with nucleotidebase, up to and including all nucleotide bases in a particular targetpolynucleotide. In some embodiments, a nucleotide base may interact withadjacent nucleotide bases on the same nucleic acid strand by hydrophobicstacking. Universal nucleotides include, but are not limited to,deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyriltriphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate(dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxylmPytriphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), ordeoxypropynyl-7-azaindole triphosphate (dP7AITP). Further examples ofsuch universal bases can be found, inter alia, in Published U.S.application Ser. No. 10/290672, and U.S. Pat. No. 6,433,134.

As used herein, the terms “polynucleotide” and “oligonucleotide” areused interchangeably and mean single-stranded and double-strandedpolymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA)and ribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, e.g., 3′-5′ and 2′-5′, inverted linkages, e.g., 3′-3′ and5′-5′, branched structures, or internucleotide analogs. Polynucleotideshave associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+,Na+, and the like. A polynucleotide may be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. Polynucleotides may be comprised of internucleotide, nucleobaseand/or sugar analogs. Polynucleotides typically range in size from a fewmonomeric units, e.g., 3-40 when they are more commonly frequentlyreferred to in the art as oligonucleotides, to several thousands ofmonomeric nucleotide units. Unless denoted otherwise, whenever apolynucleotide sequence is represented, it will be understood that thenucleotides are in 5′ to 3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytosine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, unless otherwise noted.

As used herein, “nucleobase” means those naturally occurring and thosenon-naturally occurring heterocyclic moieties commonly known to thosewho utilize nucleic acid technology or utilize peptide nucleic acidtechnology to thereby generate polymers that can sequence specificallybind to nucleic acids. Non-limiting examples of suitable nucleobasesinclude: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil,2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitablenucleobase include those nucleobases illustrated in FIGS. 2(A) and 2(B)of Buchardt et al. (WO92/20702 or WO92/20703).

As used herein, “nucleobase sequence” means any segment, or aggregate oftwo or more segments (e.g. the aggregate nucleobase sequence of two ormore oligomer blocks), of a polymer that comprises nucleobase-containingsubunits. Non-limiting examples of suitable polymers or polymerssegments include oligodeoxynucleotides (e.g., DNA), oligoribonucleotides(e.g., RNA), peptide nucleic acids (PNA), PNA chimeras, PNA combinationoligomers, nucleic acid analogs and/or nucleic acid mimics.

As used herein, “polynucleobase strand” means a complete single polymerstrand comprising nucleobase subunits. For example, a single nucleicacid strand of a double stranded nucleic acid is a polynucleobasestrand.

As used herein, “nucleic acid” is a nucleobase sequence-containingpolymer, or polymer segment, having a backbone formed from nucleotides,or analogs thereof.

Preferred nucleic acids are DNA and RNA.

As used herein, nucleic acids may also refer to “peptide nucleic acid”or “PNA” means any oligomer or polymer segment (e.g., block oligomer)comprising two or more PNA subunits (residues), but not nucleic acidsubunits (or analogs thereof), including, but not limited to, any of theoligomer or polymer segments referred to or claimed as peptide nucleicacids in U.S. Pat. Nos. 5,539,082; 5,527,675; 5,623,049; 5,714,331;5,718,262; 5,736,336; 5,773,571; 5,766,855; 5,786,461; 5,837,459;5,891,625; 5,972,610; 5,986,053; and 6,107,470; all of which are hereinincorporated by reference. The term “peptide nucleic acid” or “PNA”shall also apply to any oligomer or polymer segment comprising two ormore subunits of those nucleic acid mimics described in the followingpublications: Lagriffoul et al., Bioorganic & Medicinal ChemistryLetters, 4:1081-1082 (1994); Petersen et al., Bioorganic & MedicinalChemistry Letters, 6:793-796 (1996); Diderichsen et al., Tett. Lett.37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7:637-627(1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690 (1997); Krotzet al., Tett. Lett. 36:6941-6944 (1995); Lagriffoul et al., Bioorg. Med.Chem. Lett. 4:1081-1082 (1994); Diederichsen, U., Bioorganic & MedicinalChemistry Letters, 7:1743-1746 (1997); Lowe et al., J. Chem. Soc. PerkinTrans. 1, (1997)1:539-546; Lowe et al., J. Chem. Soc. Perkin Trans.11:547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560(1997); Howarth et al., J. Org. Chem. 62:5441-5450 (1997); Altmann, K-Het al., Bioorganic & Medicinal Chemistry Letters, 7:1119-1122 (1997);Diederichsen, U., Bioorganic & Med. Chem. Lett., 8:165-168 (1998);Diederichsen et al., Angew. Chem. Int. Ed., 37:302-305 (1998); Cantin etal., Tett. Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron,53:1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3:912-919(1997); Kumar et al., Organic Letters 3(9):1269-1272 (2001); and thePeptide-Based Nucleic Acid Mimics (PENAMS) of Shah et al. as disclosedin WO96/04000.

Lipid Particle Characteristics

Morphology. The lipid particle of the invention differs from othersimilarly constituted materials by its morphology and characterized ashaving a substantially solid core. A lipid particle having asubstantially solid core is a particle that does not have extendedaqueous regions on the interior and that has an interior that isprimarily lipid. In one embodiment, an extended region is a continuousaqueous region with a volume greater than half the particle volume. In asecond embodiment, an extended aqueous region is more than 25% of theparticle volume. The extent of internal aqueous regions may bedetermined by electron microscopy and appear as regions of low electrondensity. Further, because the interior of the solid core nanoparticle isprimarily lipid, the aqueous content of the particle (the “trappedvolume”) per lipid constituting the particle is less than that expectedfor a unilamellar bilayer lipid vesicle with the same radius. In oneembodiment, the trapped volume is less than 50% of that expected for aunilamellar bilayer vesicle with the same radius. In a secondembodiment, the trapped volume is less than 25% of that expected for aunilamellar bilayer vesicle of the same size. In a third embodiment, thetrapped volume is less than 20% of the total volume of the particle. Inone embodiment, the trapped volume per lipid is less than 2 microliterper micromole lipid. In another embodiment the trapped volume is lessthan 1 microliter per micromole lipid. In addition, while the trappedvolume per lipid increases substantially for a bilayer lipid vesicle asthe radius of the vesicle is increased, the trapped volume per lipiddoes not increase substantially as the radius of solid corenanoparticles is increased. In one embodiment, the trapped volume perlipid increases by less than 50% as the mean size is increased from adiameter of 20 nm to a diameter of 100 nm. In a second embodiment, thetrapped volume per lipid increases by less than 25% as the mean size isincreased from a diameter of 20 nm to a diameter of 100 nm. The trappedvolume can be measured employing a variety of techniques described inthe literature. Because solid core systems contain lipid inside theparticle, the total number of particles of a given radius generated permole of lipid is less than expected for bilayer vesicle systems. Thenumber of particles generated per mol of lipid can be measured byfluorescence techniques amongst others.

The lipid particles of the invention can also be characterized byelectron microscopy. The particles of the invention having asubstantially solid core have an electron dense core as seen by electronmicroscopy. Electron dense is defined such that area-averaged electrondensity of the interior 50% of the projected area of a solid coreparticle (as seen in a 2-D cryo EM image) is not less than x % (x=20%,40%, 60%) of the maximum electron density at the periphery of theparticle. Electron density is calculated as the absolute value of thedifference in image intensity of the region of interest from thebackground intensity in a region containing no nanoparticle.

The results presented in this work demonstrate that a microfluidicdevice containing a staggered herringbone mixer can be used to generateLNP with a variety of lipid compositions, can be used to efficientlyencapsulate OGN. There are three aspects of these results that are ofparticular interest. The first concerns the mechanisms whereby LNP andLNP OGN systems are formed using the microfluidics device, the secondthe advantages of the microfluidics approach as compared to previouslyavailable procedures and third the potential improvements that can bemade to this process.

With regard to the mechanism(s) whereby the microfluidics process allowsthe formation of LNP and LNP containing OGN, two points of interestconcern the mechanism whereby LNP of 100 nm size or smaller are formedand the mechanism whereby OGN can be encapsulated to levels approaching100%. With regard to formation of LNP the rate of mixing is clearly theimportant parameter. Rapid mixing of the ethanol-lipid solution withaqueous buffer results in an increased polarity of the medium to somecritical value where the dissolved lipids come out of solution and formbilayers. Rapid mixing results in high supersaturation of lipid unimersthroughout the mixing volume, and consequently a rapid and homogeneousnucleation of nanoparticles. Higher flow rates increase the rate ofsupersaturation and thus result in higher nucleation rates. Increasednucleation and growth of nanoparticles depletes the surrounding liquidof free lipid, limiting subsequent growth by the aggregation of freelipid. This proposed mechanism is consistent with the observation thatlower concentrations of the lipid in ethanol (reduced free lipid) resultin smaller LNP and that higher flow rates, causing a faster and morehomogeneous approach to supersaturation, lead to formation of smallerLNP.

The observation that LNP OGN systems formulated by the microfluidicstechnique exhibit OGN encapsulation efficiencies approaching 100% isdifficult to understand in the absence of further morphological andother studies. Cryo-TEM studies on the LNP OGN produced by microfluidicsshow that the interior of the LNP is electron dense, suggestive of asolid core of lipid and OGN. This would suggest an encapsulation processwhereby cationic lipid and OGN associates and serves as a nucleus forsubsequent coating by DSPC and PEG-lipid. In any event, the ability ofthe microfluidics formulation process to allow encapsulationefficiencies for antisense and siRNA OGN approaching 100% independent ofnucleic acid composition is a major advantage of the procedure.

Particle size. The lipid particle of the invention has a diameter (meanparticle diameter) from about 15 to about 300 nm. In some embodiments,the lipid particle has a diameter of about 300 nm or less, 250 nm orless, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less.In one embodiment, the lipid particle has a diameter from about 15 toabout 100 nm. These particles generally exhibit increased circulatorylifetime in vivo compared to large particles. In one embodiment, thelipid particle has a diameter from about 15 to about 50 nm. Theseparticles are capable of advantageously escaping the vascular system. Inone embodiment, the lipid particle has a diameter from about 15 to about20 nm. These particles near the limit size for particles that contain anucleic acid; such particles may include a single polynucleotide (e.g.,siRNA).

The lipid particles of the invention have a diameter from about 30 toabout 200 nm. In one embodiment, the lipid particles have a diameter ofabout 80 nm.

The lipid particles of the invention are substantially homogeneous intheir size distribution. In certain embodiments, the lipid particles ofthe invention have a mean particle diameter standard deviation of fromabout 65 to about 25%. In one embodiment, the lipid particles of theinvention have a mean particle diameter standard deviation of about 60,50, 40, 35, or 30%.

Encapsulation efficiency. The lipid particles of the invention can befurther distinguished by the encapsulation efficiency. As describedbelow, the lipid particles of the invention are prepared by a process bywhich nearly 100% of the nucleic acid used in the formation process isencapsulated in the particles. In one embodiment, the lipid particlesare prepared by a process by which from about 90 to about 95% of thenucleic acid used in the formation process is encapsulated in theparticles.

Microfluidic Methods for Making Lipid Particles

In one aspect, the invention provides a method for making lipidparticles containing a therapeutic agent. In one embodiment, the methodincludes

(a) introducing a first stream comprising a therapeutic agent (e.g.,polynucleic acid) in a first solvent into a microchannel; wherein themicrochannel has a first region adapted for flowing one or more streamsintroduced into the microchannel and a second region for mixing thecontents of the one or more streams;

(b) introducing a second stream comprising lipid particle-formingmaterials in a second solvent in the microchannel to provide first andsecond streams flowing under laminar flow conditions, wherein the lipidparticle-forming materials comprise an ionizable lipid, and wherein thefirst and second solvents are not the same;

(c) flowing the one or more first streams and the one or more secondstreams from the first region of the microchannel into the second regionof the microchannel; and

(d) mixing of the contents of the one or more first streams and the oneor more second streams flowing under laminar flow conditions in thesecond region of the microchannel to provide a third stream comprisinglipid particles with encapsulated therapeutic agents.

The contents of the first and second streams can be mixed by chaoticadvection. In one embodiment, mixing the contents of the one or morefirst streams and the one or more second streams comprises varying theconcentration or relative mixing rates of the one or more first streamsand the one or more second streams. In the above embodiment, unlikeknown methods, the method does not include a dilution after mixing.

To further stabilize the third stream containing the lipid particleswith encapsulated therapeutic agents, the method can, but need notfurther include, comprising diluting the third stream with an aqueousbuffer. In one embodiment, diluting the third stream includes flowingthe third stream and an aqueous buffer into a second mixing structure.In another embodiment, the aqueous buffer comprising lipid particleswith encapsulated therapeutic agents is dialyzed to reduce the amount ofthe second solvent.

The first stream includes a therapeutic agent in a first solvent.Suitable first solvents include solvents in which the therapeutic agentsare soluble and that are miscible with the second solvent. Suitablefirst solvents include aqueous buffers. Representative first solventsinclude citrate and acetate buffers.

The second stream includes lipid particle-forming materials in a secondsolvent. Suitable second solvents include solvents in which theionizable lipids are soluble and that are miscible with the firstsolvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran,acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, andalcohols. Representative second solvents include aqueous ethanol 90%.

The methods of the invention are distinguished from other microfluidicmixing methods in several ways. Whereas certain known methods require anequal or substantially equal proportion of aqueous and organic solvents(i.e., 1:1), the method of the invention generally utilizes a solventratio of aqueous to organic that exceeds 1:1. In certain embodiments,the solvent ratio of aqueous to organic is about 2:1. In certainembodiments, the solvent ratio of aqueous to organic is about 3:1. Incertain embodiments, the solvent ratio of aqueous to organic is about4:1. In certain other embodiments, the solvent ratio of aqueous toorganic is about 5:1, about 10:1, about 50:1, about 100:1, or greater.

The lipid particles of the invention are advantageously formed in amicrofluidic process that utilizes relatively rapid mixing and high flowrates. The rapid mixing provides lipid particles having the advantageousproperties noted above including size, homogeneity, encapsulationefficiency. Mixing rates used in the practice of the method of theinvention range from about 100 μsec to about 10 msec. Representativemixing rates include from about 1 to about 5 msec. Whereas hydrodynamicflow focusing methods operate at relatively low flow rates (e.g., 5 to100 μL/minute) with relatively low lipid volumes, the method of theinvention operates at relatively high flow rates and relatively highlipid volumes. In certain embodiments, for methods that incorporate asingle mixing region (i.e., mixer), the flow rate is about 1 mL/min. Formethods of the invention that utilize mixer arrays (e.g., 10 mixers),flow rates of 40 mL/minute are employed (for 100 mixers, flow rate 400mL/min). Thus, the methods of the invention can be readily scaled toprovide quantities of lipid particles necessary for demanding productionrequirements. Coupled with the advantageous particle size andhomogeneity and encapsulation efficiencies realized, the method of theinvention overcomes disadvantages of known microfluidic methods forproducing the lipid particles. One advantage of the methods of theinvention for making the lipid particles is that the methods arescalable, which means that the methods do not change on scaling and thatthere is excellent correspondence on scaling.

Microfluidic Devices for Making Lipid Particles

In another aspect, the invention provides devices for producing a lipidparticle encapsulating a nucleic acid. In one embodiment the deviceincludes:

(a) a first inlet for receiving a first solution comprising a nucleicacid in a first solvent;

(b) a first inlet microchannel in fluid communication with the firstinlet to provide a first stream comprising the nucleic acid in the firstsolvent;

(c) a second inlet for receiving a second solution comprising lipidparticle-forming materials in a second solvent;

(d) a second inlet microchannel in fluid communication with the secondinlet to provide a second stream comprising the lipid particle-formingmaterials in the second solvent;

(e) a third microchannel for receiving the first and second streams,wherein the third microchannel has a first region adapted for flowingthe first and second streams introduced into the microchannel underlaminar flow conditions and a second region adapted for mixing thecontents of the first and second streams to provide a third streamcomprising lipid particles with encapsulated nucleic acid.

In one embodiment, the device further includes means for diluting thethird stream to provide a diluted stream comprising stabilized lipidparticles with encapsulated therapeutic agent.

The device of the invention is a microfluidic device including one ormore microchannels (i.e., a channel having its greatest dimension lessthan 1 millimeter). In one embodiment, the microchannel has ahydrodynamic diameter from about 20 to about 300 μm. As noted above, themicrochannel has two regions: a first region for receiving and flowingat least two streams (e.g., one or more first streams and one or moresecond streams) under laminar flow conditions. The contents of the firstand second streams are mixed in the microchannel's second region. In oneembodiment, the second region of the microchannel has a principal flowdirection and one or more surfaces having at least one groove orprotrusion defined therein, the groove or protrusion having anorientation that forms an angle with the principal direction (e.g., astaggered herringbone mixer), as described in U.S. ApplicationPublication No. 2004/0262223, expressly incorporated herein by referencein its entirety. In one embodiment, the second region of themicrochannel comprises bas-relief structures. To achieve maximal mixingrates, it is advantageous to avoid undue fluidic resistance prior to themixing region. Thus, one embodiment of the invention is a device inwhich non-microfluidic channels, having dimensions greater than 1000microns, are used to deliver the fluids to a single mixing channel.

In other aspects of the invention, the first and second streams aremixed with other micromixers. Suitable micromixers include dropletmixers, T-mixers, zigzag mixers, multilaminate mixers, or other activemixers.

Mixing of the first and second streams can also be accomplished withmeans for varying the concentration and relative flow rates of the firstand second streams.

In another embodiment, the device for producing a lipid particleencapsulating a nucleic acid includes microchannel for receiving thefirst and second streams, wherein the microchannel has a first regionadapted for flowing the first and second streams introduced into themicrochannel under laminar flow conditions and a second region adaptedfor mixing the contents of the first and second streams to provide athird stream comprising lipid particles with encapsulated therapeuticagent. In this embodiment, the first and second stream are introducedinto the microchannel by means other than first and second microchannelsas noted above.

To achieve maximal mixing rates it is advantageous to avoid unduefluidic resistance prior to the mixing region. Thus one embodiment ofthe invention is a device in which non-microfluidic channels, havingdimensions greater than 1000 microns, are used to deliver fluids to asingle mixing channel This device for producing a lipid particleencapsulating a nucleic acid includes:

(a) a single inlet microchannel for receiving both a first solutioncomprising a nucleic acid in a first solvent and a second solutioncomprising lipid particle-forming materials in a second solvent;

(b) a second region adapted for mixing the contents of the first andsecond streams to provide a third stream comprising lipid particles withencapsulated nucleic acid.

In such an embodiment, the first and second streams are introduced intothe microchannel by a single inlet or by one or two channels not havingmicro-dimensions, for example, a channel or channels having dimensionsgreater than 1000 μm (e.g., 1500 or 2000 μm or larger). These channelsmay be introduced to the inlet microchannel using adjacent or concentricmacrosized channels.

FIG. 1 is a schematic illustration of a representative fluidic device ofthe invention. Referring to FIG. 1, device 100 includes Region A forreceiving a first stream comprising a therapeutic agent in a firstsolvent and Region B for receiving a stream comprising lipidparticle-forming materials in a second solvent. First and second streamsare introduced into Region C flowing under laminal flow conditions, toRegion D where rapid mixing occurs, and then to Region E where the finalproduct, lipid particles containing therapeutic agent, exit the device.

FIG. 2 is a schematic illustration of a representative fluidic device ofthe invention that is an elaboration of the device and methodillustrates in FIG. 1. Referring to FIG. 2, device 200 includes Region Afor receiving a first stream comprising a therapeutic agent in a firstsolvent into a microchannel, wherein the microchannel has a first regionadapted for flowing one or more streams (A-a) are introduced (A-b) andmixed (A-c); Region B for receiving a second stream comprising lipidparticle-forming materials in a second solvent, wherein the microchannelhas a first region adapted for flowing one or more streams (B-a) areintroduced (B-b) and mixed (B-c); Region C introduces the flows ofRegion A and Region B under laminal flow conditions (C-a) and rapidlymixed (C-b); and Region D, where the formulation is ready for furtherprocessing such as dilution, pH adjustment, or other events required fornanoparticle synthesis, or where the final product, lipid particlescontaining therapeutic agent, exit the device

FIG. 3 is a schematic illustration of a representative fluidic device ofthe invention that is an elaboration of the device and methodillustrated in FIG. 2. Referring to FIG. 3, device 300 includes Region Afor receiving a first stream comprising a therapeutic agent in a firstsolvent into a microchannel, wherein the microchannel has a first regionadapted for flowing one or more streams (A-a) are introduced (A-b) andmixed (A-c); Region B for receiving a second stream comprising lipidparticle-forming materials in a second solvent, wherein the microchannelhas a first region adapted for flowing one or more streams (B-a) areintroduced (B-b) and mixed (B-c); Region C introduces the flows ofRegion A and Region B under laminal flow conditions (C-a) and rapidlymixed (C-b); Region D for receiving a third stream comprising of anynumber of materials including further particle-forming materials,dilution, pH adjustments, or other events required for nanoparticlesynthesis; Region E introduces the flows of Region C and Region D underlaminal flow conditions (E-a) and rapidly mixed (E-b); Region F, wherethe formulation is ready for further processing like dilution, pHadjustments, or other events required for nanoparticle synthesis, orwhere the final product, lipid particles containing therapeutic agent,exit the device.

FIG. 4 is a schematic illustration of another representative fluidicdevice (400) of the invention. FIG. 5 is a schematic illustration of arepresentative array of the representative fluidic device illustrated inFIG. 4.

FIG. 6 is a schematic illustration of another representative fluidicdevice (600) of the invention. Referring to FIG. 6, device 600 includesmixing channels 610 a, 610 b, and 610 c. FIG. 7 is a schematicillustration of a representative array of the representative fluidicdevice illustrated in FIG. 6.

The formation of nanoparticles on microfluidic devices is limited by thereagent volumes that participate in the mixing event and the limitedbackpressure that devices can withstand before leakage occurs. Singleelements of the herringbone or multilaminate mixer achieve a 100-1000fold increase in flow rate compared to droplet or flow focusingapproaches. In order to achieve production scale throughput, multiplemixer elements can be arrayed. In one embodiment each reagent isdistributed to the individual mixer elements using a low impedance buschannel. If the impedance of the bus channel is negligible compared tothe impedance of the mixer element, the individual flow rates at theinlet of each mixer are identical. As multiple mixer elements areoperated in parallel, the impedance of the system decreases resulting ina higher volumetric throughput. This has the advantage that the mixingcharacteristics that are observed using a single mixer element can bemaintained in a mixer array. In one embodiment, mixing in each mixerarray element is achieved by introducing multiple streams into amicrochannel. In this case the streams will mix by diffusion. The widthof the streamlines may be varied by controlling the relative flow ratesthrough the injection channels (e.g., by adjusting the dimensions ofthese channels (FIG. 5). In another embodiment, mixing is achieved bychaotic advection (staggered herringbone mixer, SHM). As shown in FIG.7, each mixer element of the array may consist of a series of mixers. Byadding elements to each array subset additional functionalities can beintegrated in-line on the microfluidic device. Such functionalities mayinclude on-chip dilution, dialysis, pH adjustments or other events thatrequire interlaced streamlines, streams sharing the same channel orstreams that are separated from each other by a porous material. In oneembodiment, 10 mM POPC is dissolved in 100% ethanol and mixed withphosphate buffered saline (PBS), pH 7.4 in the first mixer element ofeach array subset. The LNPs that are formed after mixing are stabilizedby diluting the mixture by a factor of 2 with PBS.

Table 1 compares the size distribution of particles formed on a singlemixer and a mixer array consisting of ten individual mixers. The totalflow rate through a single mixer may be 4 ml/min with a mixing ratio of50:50 at each intersection. The volumetric throughput can be increasedten-fold by operating ten mixers in parallel resulting in a totalvolumetric flow rate of 40 ml/min While the throughput of the array isamenable to production scale synthesis, LNP dimensions are maintained.

TABLE 1 Size distribution of particles formed on a single mixer and amixer array consisting of ten individual mixers. Diameter (nm) Std.Deviation (nm) Chi squared Single Mixer Intensity 73.0 37.8 1.58 Volume62.8 32.5 Number 25.7 13.3 Mixer Array Intensity 72.1 35.1 1.08 Volume62.7 32.4 Number 27.0 13.7

Any combination of any number of parallel reagent inlets, sequentialmixing chambers, and branching architectures can be used to optimize thenanoparticle formulation process. This has the advantage that differentformulation process can be precisely controlled and multiple steps ofthe nanoparticle formulation process can be integrated. Examplesinclude, but are not limited to, the following: (a) the introduction oftwo or more inlets consisting of combinations of distinct (FIG. 8) orthe same (FIG. 9) reagents to allow for the independent input control(uses include independent control of the flow rates of the inputreagents, varying the ratios between input reagents, and others; (b) twoor more mixers in sequence to allow for the sequential addition ofnanoparticle reagents or formulation processing steps (FIG. 10) (usesinclude the addition of input reagents in sequence for the controlledbottom up assembly of nanoparticles, the integration of formulationprocesses like dilution, pH adjustments, or other events required fornanoparticle synthesis, and others; or (c) any combination of inputs,mixing chambers, and branching architectures, FIG. 11 and FIG. 12illustrate two step and three step mixers with a varying number ofparallel reagent inputs and branching microfluidic structures (usesinclude the integration of multiple steps of the formulation processthat includes on chip mixing of nanoparticle reagents, nanoparticlenucleation and growth, on-chip dilution, dialysis, pH adjustments orother events required for nanoparticle synthesis.

FIG. 13 is a schematic illustration of a representative fluidic deviceof the invention having a multilaminate mixer. Referring to FIG. 13,device 1300 includes mixing channels 1310. FIG. 14 is a close-up view ofthe multilaminate mixer illustrated in FIG. 14.

As described above, methods of making lipid micro/nanoparticles havebeen conventionally “top down” approaches where larger structures areformed by dispersion of lipids in water, followed by disruption of themultilamellar vesicles (micron size range) through polycarbonate filterswith a pore size such as 100 nm or alternatively, using tip sonication.

One aspect lacking in such batch processes is the ability to preciselycontrol the structure and assembly of each lipid mixture constituent.This is especially important if certain constituents are easily degradedif exposed to their external environment, or if certain ligands mustreside on the exterior of the particle for targeting purposes. Forexample, with therapeutics it may be important to first produce aparticle that has a net positive or negative surface charge to associatea certain therapeutic drug. Further processing may then be needed tocomplete the assembly, by encapsulating such a particle with other lipidmaterial or to modify its surface characteristics. This may for exampleinclude addition of lipids to produce a net neutral particle, oraddition of targeting molecules that must reside on the exterior of theparticle for functional purposes.

In one embodiment, the method for making lipid nanoparticles includessequential assembly and growth of lipid nanoparticles through chargeassociation, and further, can provide encapsulation of therapeutic smallinterfering RNA (siRNA). This method can be used to completely alter thesurface charge characteristics from net positive to net negative andvice versa.

A lipid nanoparticle encapsulating siRNA (−ive) was prepared with acharge ratio of about 2 (+ive/−ive). The lipids included 90 mol %DLin-KC2-DMA (+ive) and 10 mol % PEG-c-DMA. The resulting particle was23 nm in diameter (FIG. 28A) and had a positive zeta potential of about7 mV (FIG. 28B). The anionic lipid was then incorporation in 4-foldexcess to the cationic lipid via microfluidic mixing. This led to anincrease in particle size to 33 nm and a shift to a negative zetapotential of −14 mV. Further incorporation of additional cationic lipid(in 4-fold excess to the previous DOPS) and then incorporation of DOPSled to a continued increase in particle size and alteration between netpositive and net negative zeta potentials.

The results were obtained by mixing in a single microfluidic mixer,recovered, and then re-injected into the micromixer to add the nextlipid component. However, a single microfluidic device could be designedto produce such particles in a continuous manner (FIG. 29).

The following devices minimize the fluidic impedances and theinteraction between the lipid and aqueous fluids prior to entering themicromixer.

FIG. 30 is a schematic representation of a representative device 3000and method of the invention. Referring to FIG. 30, device 3000 includesRegion A, where a first stream comprising a polynucleic acid in a firstsolvent into a channel of large width (>2 mm) and Region B, where astream comprising lipid particle-forming materials in a second solventinto a channel of large width (>2 mm). The streams are introduced intoRegion C where rapid mixing occurs in a micromixer and then ultimatelyto Region D, the final product.

FIG. 31 is a schematic representation of a representative device andmethod of the invention. Referring to FIG. 31, device 3100 includesRegion A, where a first stream comprising a polynucleic acid in a firstsolvent into a channel of large width (>2 mm) and Region B, where astream comprising lipid particle-forming materials in a second solventinto a channel of large width (>2 mm). The streams are introduced intoRegion C where rapid mixing occurs in a micromixer and then ultimatelyto Region D, the final product.

FIG. 32 is a schematic representation of a representative device andmethod of the invention. Referring to FIG. 32, device 3200 includesRegion A, where a first stream comprising a polynucleic acid in a firstsolvent into a channel of large width (>2 mm); Region B, where a secondstream comprising the first solvent to act as sheath fluid for the flowof Region A; Region C, where a stream comprising lipid particle-formingmaterials in a second solvent into a channel of large width (>2 mm); andRegion D, where a second stream comprising the second solvent to act assheath fluid for the flow of Region C. The streams are introduced intoRegion E where rapid mixing occurs in a micromixer and then ultimatelyto Region F, the final product. The dotted lines represent fluidicinterfaces.

FIG. 33 is a schematic representation of a representative device andmethod of the invention. Referring to FIG. 33, device 3300 includesRegion A, where a first stream comprising an polynucleic acid in a firstsolvent and a second stream comprising lipid particle-forming materialsin a second solvent are flown concentrically into a channel, areintroduced into Region B where rapid mixing occurs in a micromixer andthen ultimately to Region C where we have the final product. The twofluids in Region A may be separated by a physical barrier or by sheathfluid as demonstrated in the cross-sectional view.

Method for Delivering Therapeutic Agents Using Lipid Particles

The lipid particles of the present invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using nucleic acid-lipid particles of the present invention.The methods and compositions may be readily adapted for the delivery ofany suitable therapeutic agent for the treatment of any disease ordisorder that would benefit from such treatment.

In certain embodiments, the present invention provides methods forintroducing a nucleic acid into a cell. Preferred nucleic acids forintroduction into cells are siRNA, miRNA, immune-stimulatingoligonucleotides, plasmids, antisense and ribozymes. These methods maybe carried out by contacting the particles or compositions of thepresent invention with the cells for a period of time sufficient forintracellular delivery to occur.

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively, applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products. Methods of the present invention maybe practiced in vitro, ex vivo, or in vivo. For example, thecompositions of the present invention can also be used for deliver ofnucleic acids to cells in vivo, using methods which are known to thoseof skill in the art.

The delivery of siRNA by a lipid particle of the invention and itseffectiveness in silencing gene expression is described below.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally (e.g., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly).In particular embodiments, the pharmaceutical compositions areadministered intravenously or intraperitoneally by a bolus injection.Other routes of administration include topical (skin, eyes, mucusmembranes), oral, pulmonary, intranasal, sublingual, rectal, andvaginal.

In one embodiment, the present invention provides a method of modulatingthe expression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of thepresent invention that is associated with a nucleic acid capable ofmodulating the expression of a target polynucleotide or polypeptide. Asused herein, the term “modulating” refers to altering the expression ofa target polynucleotide or polypeptide. Modulating can mean increasingor enhancing, or it can mean decreasing or reducing.

In related embodiments, the present invention provides a method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is selected from an siRNA, a microRNA, an antisenseoligonucleotide, and a plasmid capable of expressing an siRNA, amicroRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a lipid particle of the invention and a pharmaceuticallyacceptable carrier or diluent. Representative pharmaceuticallyacceptable carriers or diluents include solutions for intravenousinjection (e.g., saline or dextrose). The composition can take the formof a cream, ointment, gel, suspension, or emulsion.

The following is a description of a representative LNP system, deviceand method for making the LNP system, and method for using a LNP fordelivering therapeutic agents.

Rapid microfluidic mixing allows production of monodisperse lipidnanoparticles. Formulation of lipid nanoparticles was performed byrapidly mixing a lipid-ethanol solution with an aqueous buffer inside amicrofluidic mixer (FIG. 15B) designed to induce chaotic advection andprovide a controlled mixing environment at intermediate Reynolds number(24<Re<240). The microfluidic channel contains herringbones thatgenerate a chaotic flow by changing the orientation of herringbonestructures between half cycles, causing a periodic change in the centersof local rotational and extensional flow.

To determine mixing performance inside the device, the pH sensitivity offluorescein was used where two 10 μM fluorescein streams were mixed, onefluorescent at pH 8.88 and the other non-fluorescent at pH 5.15. Thechannel length required for mixing to occur (extent of mixing >95%) wasfound to be between 0.8 cm and 1.0 cm. This resulted in mixing times ofapproximately 45 ms, 10 ms, and 5 ms and 3 ms for flow rates of 0.1ml/min, 0.4 ml/min, 0.7 ml/min and 1.0 ml/min, respectively. The smalldifference in mixing length is expected in a chaotic flow, which growsonly logarithmically with Péclet number (Pe=Ul/D where U is the fluidvelocity, 1 is the cross-sectional channel length, and D is thediffusivity of the molecule).

The following representative formulations include an ionizable cationiclipid, DLin-KC2-DMA, having an apparent pKa of 6.7 rendering the lipidsuitable for encapsulation of siRNA at low pH and providing a nearneutral cationic surface charge density at physiological pH. Using thisLNP-siRNA scheme as a model system, the effect of flow rate on LNPformation was determined. As the mixing time dramatically decreases withincreased flow rate, the speed at which lipids are introduced into theaqueous phase was expected to influence their final size and dispersity.Using identical flow rates, from 0.1 ml/min to 1 ml/min per channel,FIG. 16B shows the mean particle diameter of LNP-siRNA systems producedby the microfluidic mixer. The buffer contained siRNA to yield asiRNA/total lipid ratio of 0.06 (wt/wt) and the LNP mixture was diluteddirectly into buffer to reduce ethanol content to approximately 22 vol%. Particle size decreased significantly when increasing total flow ratefrom 0.2 ml/min to 2 ml/min. Particle size was largest under a flow rateof 0.2 ml/min and the LNP reached a limit size of approximately 40 nm asdetermined from the number-weighted particle diameter. Alternatively,the mixing time was also adjusted by changing the ratio of the ethanoland aqueous streams. Increasing the flow rate of the aqueous stream ineffect provides a quicker dilution of the lipids with the aqueousstream. With the lipid-ethanol stream kept constant at 0.5 ml/min, anincrease in the aqueous flow rate resulted in a decrease in particlesize (FIG. 16C). The substantial drop in particle size, from about 70 nmto 35 nm, with a three-fold increase in the aqueous flow rate highlightsthe importance in rapidly reducing the ethanol content.

Because these LNP are expected to form spontaneously as the lipidsencounter a more aqueous environment, it was also important to explorethe effect of lipid concentration. As lipid concentration is increased,the amount of lipids available to incorporate into a LNP would beexpected to increase or otherwise form additional particles. This wasmonitored as the lipid concentration was increased from 10 mM to 50 mMin the ethanol stream. An increase in mean particle diameter from about40 nm to 70 nm was observed following this increase in lipidconcentration (FIG. 17).

Rapid microfluidic mixing provides a broad formulation range ofLNP-siRNA systems. While recent improvements of the cationic lipid haveadvanced LNP potency several fold, it has also become apparent thatfurther improvements can be provided via optimization of the LNPcomposition. In particular, it can influence their bilayer-destabilizingcapacity and endosomolytic potential or may influence their circulationbehavior at physiological pH. For example, formulations with lessPEG-lipid and increased cationic lipid have shown dramatic improvementsin in vivo efficacy of LNP systems targeting liver hepatocytes. This wasobserved in a recent report for a mouse Factor VII model, which provideda further five-fold reduction in ED50 in the optimized LNP. Although thePEG-lipid is necessary for particulate stability, it can also diminishthe membrane-destabilizing property of these LNP systems. With thepreformed vesicle (PFV) method, difficulties have been encountered whenattempting to produce LNP systems with less than 5 mol % PEG-lipid; thisis presumably due to less PEG content on the exterior of the vesicleswhich increases fusion between LNPs. Further, the incubation stepnecessary for reorganization of preformed lipid particles andencapsulation of siRNA requires ethanol solutions in the range of 30%(v/v). This increased lipid fluidity can promote instability and lead toadditional aggregation and fusion of the preformed lipid particles.

Using PEG-c-DMA, the ability of the microfluidic (MF) method (fastmixing times and short residence prior to dilution of the LNP below 25%ethanol (v/v)) to produce LNP-siRNA systems with varying PEG-lipidcontent was explored. An initial composition of DLin-KC2-DMA, DSPC,cholesterol, and PEG-c-DMA (40: 11.5: 38.5: 10 mol/mol) was used with asiRNA/total lipid ratio of 0.06 (wt/wt). Additional cholesterol was usedto compensate for the decreased amount of PEG-c-DMA. Titration ofPEG-c-DMA to 2 mol % led to only a minor increase in particle size usingthe microfluidic approach. Further decrease to 1 mol % PEG led to anincrease in diameter from about 20 nm to about 40 nm (FIG. 18A). Incontrast, the mean particle diameter using the PFV method showed aconstant increase in particle diameter, from 20 nm to 70 nm, asPEG-lipid content was decreased to 1 mol %. In addition to producing LNPwith low amounts of PEG-lipid, it is of interest to be able to vary theamount of cationic lipid. As DLin-KC2-DMA was increased from 40 mol % to70 mol %, a general increase in particle size was observed, from about40 nm to 70 nm, for those produced by the microfluidic approach (FIG.18B).

Self assembly in a microfluidic device can produce LNP with nearcomplete encapsulation. In producing LNP-siRNA systems, a robust processnecessarily will provide high percent encapsulation of the OGN product.siRNA encapsulation was evaluated by varying the siRNA/total lipid ratiofrom 0.01 to 0.2 (wt/wt) using the LNP-siRNA formulation with 1 mol %PEG. LNP formulations achieved percent encapsulation approaching 100percent over this range (FIG. 19). Upon reaching a siRNA/total lipidratio of 0.21 (wt/wt), corresponding to a charge balance between thecationic lipid and anionic siRNA (N/P=1), encapsulation was observed todiminish (data not shown). This later trend was expected due toinsufficient cationic charge required to complex the siRNA andencapsulate in the LNP.

Morphology. LNP produced by the microfluidic and preformed vesiclemethods were visualized with cryo-TEM. Particle sizes of the LNP weresimilar to that measured by dynamic light scattering. LNP-siRNA systemscontaining DLin-KC2-DMA/DSPC/Cholesterol/PEG-c-DOMG at 40/11.5/47.5/1mol % with siRNA-to-lipid ratio of 0.06 wt/wt are shown in FIG. 20A. Inaddition, empty LNP samples of the same composition are shown in FIG.20B. The particles produced are predominately spherical and homogeneousin size. LNP formulated with the preformed approach and of identicalcomposition was also imaged. These shared similar features with themicrofluidic LNP, though other features such as coffee-bean structureswere also observed. These LNP were also larger in size, as expected fromthe dynamic light scattering results.

LNP siRNA systems produced by microfluidics can be highly potent genesilencing agents in vivo. The ability of LNP siRNA systems to inducegene silencing in vivo following i.v. injection was investigated usingthe mouse Factor VII model. Formulations containingDlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DOMG with a siRNA-to-lipid ratio of0.06 (w/w) were created using the microfluidic approach. Administrationof the LNP-siRNA was by tail vein injection. The cationic lipid,DLin-KC2-DMA, was varied from 30 mol % to 60 mol % while keeping theDSPC-to-Cholesterol ratio at 0.2 wt/wt. Increasing the cationic lipidcontent in the LNP resulted in a progressive improvement in FVIIsilencing. The best performing LNP contained 60 mol % DLin-KC2-DMA,resulting in an effective dose for 50% FVII silencing at about 0.03mg/kg (FIG. 21). It is interesting to note that further increase to 70mol % led to no observable improvement in efficacy over the 60 mol %Dlin-KC2-DMA LNP.

The results demonstrate that a microfluidic device containing astaggered herringbone mixer can be used to generate LNP with a varietyof lipid compositions, can be used to efficiently encapsulate OGN suchas siRNA and that the LNP siRNA systems produced exhibit excellent genesilencing capabilities both in vitro and in vivo.

The microfluidics device and system of the invention allow for theformation of LNP and LNP containing OGN of 100 nm size or smaller andprovide OGN encapsulation 100%. With regard to formation of LNP, therate and ratio of mixing are clearly the important parameters. Rapidmixing of the ethanol-lipid solution with aqueous buffer results in anincreased polarity of the medium that reduces the solubility ofdissolved lipids, causing them to precipitate out of solution and formnanoparticles. Rapid mixing causes the solution to quickly achieve astate of high supersaturation of lipid unimers throughout the entiremixing volume, resulting in the rapid and homogeneous nucleation ofnanoparticles. Increased nucleation and growth of nanoparticles depletesthe surrounding liquid of free lipid, thereby limiting subsequent growthby the aggregation of free lipid. This proposed mechanism is consistentwith the observation that lower concentrations of the lipid in ethanol(reduced free lipid) result in smaller LNP (see FIG. 17), that higherflow rates, causing a faster and more homogeneous approach tosupersaturation, lead to formation of smaller LNP, and that increasingthe relative ratio of the aqueous to organic solvent components alsoresults in smaller particles (FIG. 17).

LNP OGN systems of the invention formulated by the microfluidics methodexhibit OGN encapsulation efficiencies approaching 100%. Previouscryo-TEM studies using the PFV technique for antisense OGN have revealedthe presence of small multilamellar vesicles leading to the possibilitythat encapsulation involves OGN adsorption to a preformed vesicle whichserves as a nucleation point for association with additional preformedvesicles that wrap around the original vesicle. In contrast, cryo-TEMstudies of the LNP OGN produced by the microfluidics method show thatthe majority of the LNP systems are “solid core” structures and suggestthat a different mechanism of OGN encapsulation is operative. Inparticular, these structures are consistent with the association ofsiRNA with cationic lipid monomers prior to or simultaneously withnanoparticle assembly. The ability of the microfluidics method tofacilitate encapsulation efficiencies for antisense and siRNA OGNapproaching 100% independent of nucleic acid composition is a majoradvantage over previously reported methods.

The microfluidics method provides advantages over three alternative LNPsynthesis techniques including the classical extrusion procedure forproducing LNP, the preformed vesicle method, and the spontaneous vesicleformation methods for OGN encapsulation. The microfluidics methodprovides LNP in the 100 nm size range or smaller and, when cationiclipid is present, allows LNP to be formed with low levels of stabilizingPEG-lipid. Disadvantages of the microfluidics method relate to the needto remove ethanol after preparation, the fact that certain lipids arerelatively insoluble in ethanol, and potential scalability issues. Themicrofluidics method offers advantages in encapsulation efficiencies,the use of high cationic lipid contents and low PEG-lipid levels thatare difficult to employ using the PFV process, removal of the need togenerate preformed vesicles, and the ability to produce small scalebatches using as little as 150 μg of oligonucleotide with little lossdue to the small dead volume (1 μl) of the apparatus.

Advantages of the microfluidics method as compared to the SVF “T tube”procedure for generating LNP systems loaded with OGN are similar tothose indicated for the PFV process, with the exception that preformedvesicles are not required. The aperture of the T tube is approximately1.5 mm in diameter, requiring high flow rates (>1 ml/s) to achieve thevelocities required for rapid mixing to occur. The micromixer allows LNPOGN formulation to occur under well defined, reproducible conditions atmuch lower flow rates and reduced losses due to dead volumes, allowingmore straightforward preparation of small-scale batches for LNPoptimization and in vitro testing.

LNP OGN systems can be scaled up. Although a device that has a maximumflow rate of 1 ml/min may be insufficient, a single microfluidics chipmay contain 10 or more micromixers in to achieve total flow rates ofabout 10 mL/min. Given the relatively inexpensive nature of thistechnology it is practical that a number of such chips to be used inparallel, potentially allowing flow rates of 100 ml/min or higher from asingle bench-top instrument. Furthermore, upstream fluid handling couldeasily be incorporated into such a device to allow for preciseprogrammable formulations from multiple components, a feature that wouldbe highly advantageous in the screening and optimization of synthesisformulations and parameters.

Solid Core LNP

Certain models of LNP siRNA formulations suggest a bilayer vesiclestructure of the LNP with siRNA on the inside in an aqueous interior.However, a number of observations suggest that such models areincorrect, at least for LNP siRNA systems generated by the microfluidicmixing approach. For example, cryo-electron microscopy of LNP siRNAsystems produced by microfluidic mixing indicates the presence ofelectron-dense cores rather than the aqueous cores consistent withvesicular structure. As noted above, formulation of LNP siRNA systemscan routinely result in siRNA encapsulation efficiencies approaching100%, an observation that is not consistent with bilayer structureswhere maximum encapsulation efficiencies of 50% might be expected.

The structure of LNP siRNA systems was evaluated employing a variety ofphysical and enzymatic assays. The results obtained indicate that theseLNP siRNA systems have a solid core interior of consisting of siRNAmonomers complexed with cationic lipid as well as lipid organized ininverted micellar or related structures.

LNP systems exhibit electron dense solid core structure as indicated bycryo EM in the presence and absence of encapsulated siRNA. LNP systemsproduced by microfluidic mixing exhibit electron dense cores asvisualized by cryo EM, consistent with a solid cores, in contrast to theaqueous core structures suggested for LNP siRNA systems created byalternative methods. This was confirmed as shown in FIG. 22A for an LNPsiRNA formulation consisting of DLin-KC2-DMA/DSPC/Chol/PEG-lipid(40/11.5/47.5/1; mol/mol) containing siRNA at a 0.06 siRNA/lipid (wt/wt)content, which corresponds to a negative charge (on the siRNA) topositive charge (on the fully protonated cationic lipid) N/P ratio of 4.As a result approximately 75% of the cationic lipid is not complexed tosiRNA in the LNP. The solid core electron dense structure contrasts withthe less dense interior of a vesicle system composed of POPC (FIG. 22B)and is visually similar to the electron dense interior of aPOPC/triolein (POPC/TO) LNP (FIG. 22C). POPC/TO LNP produced bymicrofluidic mixing consist of a hydrophobic core of TO surrounded by amonolayer of POPC.

An interesting feature of FIG. 22A is that 75% of the ionizable cationiclipid is not complexed to siRNA, but the LNP siRNA particle as a wholeexhibits a solid core interior. This suggests that the cationic lipidmay contribute to the solid core interior even when it is not complexedto siRNA. LNP systems with the same lipid composition but no siRNA wereformulated employing the microfluidics process and characterized by cryoEM. As shown in FIG. 22B, the electron dense core was observed in theabsence of siRNA, indicating that ionizable cationic lipids such asDLin-KC2-DMA, possibly in combination with DSPC and cholesterol, canadopt non-lamellar electron dense structures in the LNP interior.

LNP structures exhibit limit sizes indicating that ionizable cationiclipid forms inverted micellar structures in the LNP interior. Thecontribution of the cationic lipid to the electron dense LNP core raisesthe question of what the molecular structure of such LNP systems may be.It is logical to propose that the cationic lipid, in association with acounter-ion, adopts an inverted structure such as an inverted micelle,consistent with the propensity of these lipids for inverted structuressuch as the hexagonal H_(II) phase in mixtures with anionic lipids. Inturn, this would suggest that LNP systems composed of pure cationiclipid should exhibit limit sizes with diameters in the range of 10 nm,which is essentially the thickness of two bilayers surrounding aninverted micelle interior with diameter 2-3 nm. The diameter of theaqueous channels found for phosphatidylethanolamine in the H_(II) phaseis 2.6 nm. The microfluidics formulation process provides fast mixingkinetics that drive the generation of limit size systems for LNPsystems. The limit size that could be achieved for aDLin-KC2-DMA/PEG-lipid system (90/10, mol/mol) was evaluated. As shownin FIG. 23, measurements by dynamic light scattering on these LNP formedby the microfluidic method confirm that the particle size isapproximately 10 nm in diameter, a finding that is not consistent with asignificant aqueous core or trapped volume.

A related question concerns the structure of the cationic lipid-siRNAcomplex. Again, it is logical to suppose that it consists of a distortedinverted micelle of cationic lipid surrounding the siRNAoligonucleotide. In turn, this would suggest a limit size in the rangeof 15-20 nm, assuming that the siRNA contained in this inverted micelleis surrounded by an interior monolayer of cationic lipid and then anouter monolayer of remaining lipid and that the dimensions of the siRNAare 2.6 nm in diameter and 4.8 nm in length. In order to determinewhether this is consistent with experiment, the limit size of LNP siRNAsystems consisting of DLin-KC2-DMA and PEG-lipid (90/10; mol/mol) athigh levels of siRNA corresponding to an N/P ratio of one wasdetermined. As shown in FIG. 23, the inclusion of siRNA resulted in alimit-size systems of approximately 21 nm diameter, consistent withhypothesis.

Encapsulated siRNA is immobilized in the LNP. If the siRNA is complexedto cationic lipid and localized in a solid core inside the LNP it wouldbe expected to be less mobile than if freely tumbling in the aqueousinterior of a bilayer vesicle system. The mobility of the siRNA can beprobed using ³¹P NMR techniques. In particular, it would be expectedthat limited motional averaging would be possible for complexed siRNA,leading to very broad “solid state” ³¹P NMR resonances due to the largechemical shift anisotropy of the phosphate phosphorus. Under theconditions employed, such resonances would not be detectable. If, on theother hand, the siRNA is able to freely tumble in an aqueousenvironment, rapid motional averaging would be expected to lead tonarrow, readily detectable, ³¹P NMR spectra. In order to eliminatecomplications arising from ³¹P NMR signals arising from the phospholipidphosphorus, DSPC was omitted from formulations of LNP to test thishypothesis. As shown in FIG. 24A, for LNP siRNA systems with lipidcomposition DLin-KC2-DMA/Chol/PEG-lipid (50/45/5 mol %) and containingsiRNA (0.06 siRNA/lipid; wt/wt), no ³¹P NMR signal is observable for theencapsulated siRNA, consistent with immobilization within the LNP core.If the detergent sodium dodecyl sulphate is added (1%) to solubilize theLNP and release the encapsulated siRNA then a narrow ³¹P NMR signal isdetected as shown in FIG. 24C.

Encapsulated siRNA is fully protected from degradation by external RNaseA. A test of the internalization of siRNA is that if siRNA issequestered in the LNP core they should be fully protected fromdegradation by externally added RNase. LNP siRNA systems with the lipidcomposition DLin-KC2-DMA/DSPC/Chol/PEG-lipid (40/11/44/5 mol %) wereincubated with RNase A to determine whether encapsulated siRNA could bedigested. As shown in the gel presented in FIG. 25, the free siRNA isdegraded, while the siRNA associated within the LNP particles made bythe microfluidic method is completely protected (FIG. 25 arrow). As alsoshown in FIG. 25, addition of the detergent Triton X-100 to the LNPresults in dissolution of the LNP, release of the siRNA, and degradationin the presence of RNase.

Encapsulated siRNA is complexed with internalized cationic lipid. Thesolid core of the LNP siRNA systems consists of encapsulated siRNAcomplexed to cationic lipid and the remaining lipid (cationic lipid,cholesterol and PEG-lipid) is either present in the core in invertedmicellar or similar structures, or resident on the LNP exterior. Forhigh siRNA contents, where essentially all of the cationic lipid iscomplexed with internalized siRNA, it would be expected that littlecationic lipid would be localized on the LNP exterior. A fluorescenceresonance energy transfer (FRET) assay was developed to determineexternal cationic lipid. The assay required the preparation ofnegatively charged vesicular LNP composed of dioleoylphosphatidylserine(DOPS) that contained the FRET pair, NBD-PE/Rh-PE at high(self-quenching) concentrations. The negatively charged DOPS LNP werethen incubated with LNP siRNA systems consisting ofDLin-KC2-DMA/DSPC/Chol/PEG-lipid (40/11.5/47.5/1 mol %) at pH 5.5. ThepKa of DLin-KC2-DMA is 6.7 and thus nearly all the DLin-KC2-DMA on theoutside of the LNP will be charged at pH 5.5, promoting an interactionand potentially fusion with the negatively charged DOPS LNP. Fusion isreported as an increase in the NBD-PE fluorescence at 535 nm as theNBD-PE and Rh-PE probes become diluted following lipid mixing.

As shown in FIG. 26, when the LNP systems contained no siRNA substantialfusion is observed consistent with a considerable proportion of theDLin-KC2-DMA residing on the outer monolayer of the LNP system. When theLNP systems contained siRNA at a siRNA to lipid ratio of 0.06 (wt/wt),which corresponds to a positive (cationic lipid) charge to negative(siRNA) N/P charge ratio of 4, however, fusion was considerably reduced(FIG. 26), whereas for LNP siRNA systems prepared with an N/P of 1,little or no fusion was observed, indicating that little of noDLin-KC2-DMA was present on the LNP siRNA exterior. This supports thehypothesis that high siRNA content essentially all of the cationic lipidis complexed with siRNA and sequestered in the LNP interior.

The results provide evidence that the interior of LNP siRNA systemsconsist of a solid core composed of siRNA monomers complexed to cationiclipids, as well as lipids arranged in inverted micelle or relatedstructures. These results imply a model for LNP siRNA structure, providea rationale for the high siRNA encapsulation efficiencies that can beachieved and suggest methods for manufacturing LNP siRNA systems withproperties appropriate to particular applications.

The model for LNP siRNA structure based on the results is shown in FIG.27. The model proposes that encapsulated siRNA resides in a distortedinverted micelle surrounded by cationic lipid, and that remaining lipidis organized in inverted micelles surrounding anionic counterions andalso makes up the outermost monolayer.

The model provides an understanding of how siRNA encapsulationefficiencies approaching 100% can be achieved during the microfluidicmixing formulation process. This is a major problem for siRNAencapsulation in bilayer systems because, assuming the cationic lipid isequally distributed on both sides of the bilayer, a maximum of 50% siRNAinternalization would be expected. The model points to ways in which LNPsiRNA size, composition, and surface charge may be readily modulated.With regard to size, the limit size structure is clearly one thatcontains one siRNA monomer per particle, suggesting a limit size ofapproximately 15-20 nm. Such LNP siRNA particles are readily achievedusing microfluidic method of the invention. The limit size LNP siRNAsystem consisting of a monomer of siRNA can be potentially used as abuilding block to achieve LNP siRNA systems of varying composition andsurface charge using microfluidic mixing technology. Rapid mixing ofpreformed limit size LNP siRNA with an ethanol solution containingnegatively charged lipids, for example, may be expected to result in aninteraction with excess cationic lipids to produce internal invertedmicellar core structures and a negatively charged surface.

The lipid particles of the invention described herein include (i.e.,comprise) the components recited. In certain embodiments, the particlesof the invention include the recited components and other additionalcomponents that do not affect the characteristics of the particles(i.e., the particles consist essentially of the recited components).Additional components that affect the particles' characteristics includecomponents such as additional therapeutic agents that disadvantageouslyalter or affect therapeutic profile and efficacy of the particles,additional components that disadvantageously alter or affect the abilityof the particles to solubilize the recited therapeutic agent components,and additional components that disadvantageously alter or affect theability of the particles to increase the bioavailability of the recitedtherapeutic agent components. In other embodiments, the particles of theinvention include only (i.e., consist of) the recited components.

The following examples are provided for the purpose of illustrating, notlimiting, the claimed invention.

EXAMPLES

Materials

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine Bsulfonyl) (Rh-PE) were obtained from Avanti Polar Lipids (Alabaster,Ala.). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) andcholesterol was obtained from Sigma (St Louis, Mo.). N-[(Methoxypoly(ethylene glycol)₂₀₀₀)carbamyl]-1,2-dimyristyloxlpropyl-3-amine(PEG-C-DMA) was synthesized by AlCana Technologies.2-(N-Morpholino)ethanesulfonic acid (MES) was obtained from BDH.Ammonium acetate, sodium acetate and sodium chloride were obtained fromFisher Scientific (Fair Lawn, N.J.). RNase A was obtained from AppliedBiosystems/Ambion (Austin, Tex.). Factor VII (FVII) targeting, and lowGC negative control siRNA were purchased from Invitrogen (Carlsbad,Calif.). Factor VII siRNA: (SEQ ID NO: 1) 5′-GGAUCAUCUCAAGUCUUACTT-3′(FVII sense), and (SEQ ID NO: 2) 5′-GUAAGACUUGAGAUGAUCCTT-3′ (FVIIantisense). DLin-KC2-DMA was obtained from AlCana Technologies Inc.(Vancouver, BC).

Example 1

Preparation of LNP Systems: Preformed Vesicle Method

In the example, the preparation of an LNP-siRNA system using thepreformed vesicle method is described.

LNP-siRNA systems were made using the preformed vesicle method asdepicted in FIG. 15A and as described in N. Maurer, K. F. Wong, H.Stark, L. Louie, D. McIntosh, T. Wong, P. Scherrer, S. Semple and P. R.Cullis, “Spontaneous Entrapment of Polynucleotides Upon ElectrostaticInteraction With Ethanol Destabilized Cationic Liposomes: Formation ofSmall Multilamellar Liposomes,” Biophys. J., 80:2310-2326 (2001).Cationic lipid, DSPC, cholesterol and PEG-lipid were first solubilizedin ethanol at the appropriate molar ratio. The lipid mixture was thenadded dropwise to an aqueous buffer (citrate or acetate buffer, pH 4)while vortexing to a final ethanol and lipid concentration of 30% (v/v).The hydrated lipids were then extruded five times through two stacked 80nm pore-sized filters (Nuclepore) at room temperature using a LipexExtruder (Northern Lipids, Vancouver, Canada). The siRNA (solubilized inan identical aqueous solution containing 30% ethanol) was added to thevesicle suspension while mixing. A target siRNA/lipid ratio of 0.06(wt/wt) was generally used. This mixture was incubated for 30 minutes at35° C. to allow vesicle re-organization and encapsulation of the siRNA.The ethanol was then removed and the external buffer replaced withphosphate-buffered saline (PBS) by dialysis (12-14k MW cut-off, Spectrummedical instruments) to 50 mM citrate buffer, pH 4.0 and then dialysisto PBS, pH 7.4.

Example 2

Preparation of LNP Systems: Microfluidic Staggered Herringbone Mixer

In the example, a representative LNP-siRNA system of the invention usinga microfluidic staggered herringbone mixer is described.

LNP-siRNA preparation. Oligonucleotide (siRNA) solution was prepared in25 mM acetate buffer at pH 4.0. Depending on the desiredoligonucleotide-to-lipid ratio and formulation concentration, solutionswere prepared at a target concentration of 0.3 mg/ml to 1.9 mg/ml totallipid. A lipid solution containing DLin-KC2-DMA, DSPC, cholesterol, anda PEG-lipid at the appropriate molar ratio was prepared in ethanol anddiluted with 25 mM acetate buffer to achieve an ethanol concentration of90% (v/v). FIG. 15B is a schematic illustration of the microfluidicapparatus used in this example. The device has two inlets, one for eachof the solutions prepared above, and one outlet. The microfluidic devicewas produced by soft lithography, the replica molding of microfabricatedmasters in elastomer. The device features a 200 μm wide and 79 μm highmixing channel with herringbone structures formed by 31 μm high and 50μm thick features on the roof of the channel. Fluidic connections weremade with 1/32″ I.D., 3/32″ O.D. tubing that was attached to 21G1needles for connection with syringes. 1 ml syringes were generally usedfor both inlet streams. A dual syringe pump (KD200, KD Scientific) wasused to control the flow rate through the device. The flow rate of eachstream was varied from 0.1 ml/min to 1 ml/min. The syringe pumpintroduces the two solutions into the microfluidic device (inlet a andinlet b in FIG. 15B), where they come into contact at a Y-junction.Insignificant mixing occurs under laminar flow by diffusion at thispoint, whereas the two solutions become mixed as they pass along theherringbone structures.

Mixing occurs in these structures by chaotic advection, causing thecharacteristic separation of laminate streams to become increasinglysmall, thereby promoting rapid diffusion. This mixing occurs on amillisecond time scale and results in the lipids being transferred to aprogressively more aqueous environment, reducing their solubility andresulting in the spontaneous formation of LNP. By including cationiclipids in the lipid composition, entrapment of oligonucleotide speciesis obtained through association of the positively charged lipid headgroup and negatively charged oligonucleotide. Following mixing in themicrofluidic device, the LNP mixture was generally diluted into a glassvial containing two volumes of stirred buffer. Ethanol is finallyremoved through dialysis to 50 mM citrate buffer, pH 4.0 and thendialysis to PBS, pH 7.4. Empty vesicles were similarly produced, withthe oligonucleotide absent from the buffer solution.

LNP Image Analysis. Mixing times were measured by fluorescent imaging ofthe mixing of fluorescein solutions with different pH values. Imageswere collected using an Olympus inverted confocal microscope using a 10×objective and Kalman filter mode with 2 scans per line. Twenty-fiveequally spaced slices were taken along the height of the channel andcombined to determine total intensity profiles. For each positionimaged, ten adjacent rows of pixels along the flow direction wereaveraged to obtain an intensity profile along the width of the channeland used to determine the extent of mixing. Mixing experiments wereperformed with two 10 μM fluorescein solutions supplemented with 0.5 MNaCl to suppress the formation of a liquid junction potential due to alarge difference in sodium and phosphate ion concentrations. Onesolution contained 14 mM phosphate buffer at pH 8.88, while the othercontained 1 mM phosphate buffer at pH 5.15. The increase in fluorescenceof the solution initially at pH 5.15 will overwhelm the small drop influorescence in the basic solution, resulting in an increase in totalfluorescence intensity by a factor of two. The extent of mixing wasdetermined at approximately 2.1 mm, 6.2 mm, and 10.1 mm along thechannel length using flow rates of the individual streams at 0.1 ml/min,0.4 ml/min, 0.7 ml/min and 1.0 ml/min.

LNP Characterization. Particle size was determined by dynamic lightscattering using a Nicomp model 370 Submicron Particle Sizer (ParticleSizing Systems, Santa Barbara, Calif.). Number-weighted andintensity-weighted distribution data was used. Lipid concentrations wereverified by measuring total cholesterol using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). Removal of freesiRNA was performed with VivaPureD MiniH columns (Sartorius StedimBiotech GmbH, Goettingen, Germany) The eluents were then lysed in 75%ethanol and siRNA was quantified by measuring absorbance at 260 nm.Encapsulation efficiency was determined from the ratio ofoligonucleotide before and after removal of free oligonucleotidecontent, normalized to lipid content.

LNP Cyro-Transmission Electron Microscopy. Samples were prepared byapplying 3μL of PBS containing LNP at 20-40 mg/ml total lipid to astandard electron microscopy grid with a perforated carbon film. Excessliquid was removed by blotting with a Vitrobot system (FEI, Hillsboro,Oreg.) and then plunge-freezing the LNP suspension in liquid ethane torapidly freeze the vesicles in a thin film of amorphous ice. Images weretaken under cryogenic conditions at a magnification of 29K with an AMTHR CCD side mount camera. Samples were loaded with a Gatan 70 degreecryo-transfer holder in an FEI G20 Lab6 200kV TEM under low doseconditions with an underfocus of 5-8 μm to enhance image contrast.

In vivo Activity of LNP-siRNA for FVII activity. Six to eight week old,female C57B1/6 mice were obtained from Charles River Laboratories.LNP-siRNA containing Factor VII siRNA were filtered through a 0.2 μmfilter and diluted to the required concentrations in sterile phosphatebuffered saline prior to use. The formulations were administeredintravenously via the lateral tail vein at a volume of 10 ml/kg. After24 h, animals were anaesthetized with Ketamine/Xylazine and blood wascollected by cardiac puncture. Samples were processed to serum(Microtainer Serum Separator Tubes; Becton Dickinson, N.J.) and testedimmediately or stored at −70° C. for later analysis of serum Factor VIIlevels. All procedures were performed in accordance with local, state,and federal regulations as applicable and approved by the InstitutionalAnimal Care and Use Committee (IACUC).

Serum Factor VII levels were determined using the colorimetric BiophenVII assay kit (Anaira). Control serum was pooled and serially diluted(200%-3.125%) to produce a calibration curve for calculation of FVIIlevels in treated animals Appropriately diluted plasma samples fromtreated animals (n=3 per dosage) and a saline control group (n=4) wereanalyzed using the Biophen VII kit according to manufacturer'sinstructions. Analysis was performed in 96-well, flat bottom,non-binding polystyrene assay plates (Corning, Corning, N.Y.) andabsorbance was measured at 405 nm. Factor VII levels in treated animalswere determined from a calibration curve produced with the seriallydiluted control serum.

Example 3

LNP Systems: Solid Core

In the example, a structure of a representative LNP-siRNA system of theinvention having a solid core is described.

Preparation of lipid nanoparticles. LNP were prepared by mixing desiredvolumes of lipid stock solutions in ethanol with an aqueous phaseemploying the micro-mixer described above. For the encapsulation ofsiRNA, the desired amount of siRNA was mixed with 25 mM sodium acetatebuffer at pH 4. Equal volumes of the lipid/ethanol phase and thesiRNA/aqueous phase were combined in a micro-mixer containing aherring-bone structure to promote mixing. The ethanol content wasquickly diluted to 25% with sodium acetate buffer upon leaving themicro-mixer. The flow rate through the micro-mixing was regulated usinga dual-syringe pump (Kd Scientific). The lipid mixture then underwent a4 hour dialysis in 50 mM MES/sodium citrate buffer (pH 6.7) followed byan overnight dialysis in phosphate buffered saline (pH 7.4).

Cryo-EM. Samples were prepared by applying 3 μL of PBS containing LNP at20-40 mg/ml total lipid to a standard electron microscopy grid with aperforated carbon film. Excess liquid was removed by blotting with aVitrobot system (FEI, Hillsboro, Oreg.) and then plunge-freezing the LNPsuspension in liquid ethane to rapidly freeze the vesicles in a thinfilm of amorphous, vitreous ice. Images were taken under cryogenicconditions at a magnification of 29K with an AMT HR CCD side mountcamera. Samples were loaded with a Gatan 70 degree cryo-transfer holderin an FEI G20 Lab6 200kV TEM under low dose conditions with anunderfocus of 5-8 um to enhance image contrast.

RNase protection assay. Factor VII siRNA was encapsulated with 40%DLinKC2-DMA, 11% DSPC, 44% cholesterol and 5% PEG-c-DMA using themicrofluidics mixing method. 1 ug of siRNA was incubated with 0.05 ugRNase A (Ambion, Austin, Tex.) in 50 uL of 20 mM HEPES (pH 7.0) at 37°C. for 1 hour. At the end of the incubation, a 10 uL aliquot of thereaction mix was added to 30 uL FA dye (deionized formamide, TBE, PBS,xylene cyanol, bromophenol blue, yeast tRNA) to halt the RNase reaction.Gel electrophoresis was performed using 20% native polyacrylamide geland nucleic acids were visualized by staining with CYBR-Safe(Invitrogen, Carlsbad, Calif.).

³¹P-NMR studies. Proton decoupled ³¹P NMR spectra were obtained using aBruker AVII 400 spectrometer operating at 162 MHz. Free induction decays(FID) corresponding to about 10⁴ scans were obtained with a 15 μs,55-degree pulse with a 1 s interpulse delay and a spectral width of 64kHz. An exponential multiplication corresponding to 50 Hz of linebroadening was applied to the FID prior to Fourier transformation. Thesample temperature was regulated using a Bruker BVT 3200 temperatureunit. Measurements were performed at 25° C.

FRET membrane fusion studies. Fusion between LNP siRNA nanoparticles andanionic DOPS vesicles was assayed by a lipid mixing assay employingfluorescence resonance energy transfer. Labeled DOPS vesicles containingNBD-PE and Rh-PE (1 mol % each) were prepared by direct re-hydration oflipid film with the appropriate buffer followed by 10 extrusions througha 100 nm pore size polycarbonate membrane using the Lipex Extruder. LNPcomprised of 40% DLinKC2-DMA, 11.5% DSPC, 47.5% cholesterol, 1%PEG-c-DMA were prepared with siRNA-to-lipid ratio (D/L ratio, wt/wt) of0, 0.06 and 0.24. A D/L=0.24 represents an equimolar ratio of positive(cationic lipid) to negative (siRNA) charges (N/P=1). Lipid mixingexperiments were conducted. Labeled DOPS vesicles and unlabeled LNP weremixed at a 1:2 mol ratio into a stirring cuvette containing 2 mL of 10mM acetate, 10 mM MES, 10 mM HEPES, 130 mM NaCl equilibrated to pH 5.5.Fluorescence of NBD-PE was monitored using 465 nm excitation, and 535 nmemission using an LS-55 Perkin Elmer fluorometer using a 1×1 cm cuvetteunder continuous low speed stirring. Lipid mixing was monitored forapproximately 10 min, after which 20 μL of 10% Triton X-100 was added todisrupt all lipid vesicles, representing infinite probe dilution. Lipidmixing as a percentage of infinite probe dilution was determined usingthe equation: % lipid mixing=(F−F_(o))/(F_(max)−F_(o))×100, where F isthe fluorescence intensity at 535 nm during assay, F_(o) is the initialfluorescence intensity, and F_(max) is the maximum fluorescenceintensity at infinite probe dilution after the addition of Triton X-100.

Example 4

Sequential Assembly of Lipid Nanoparticles

In this example, a representative method of the invention, sequentialassembly, for making lipid nanoparticles is described.

The oligonucleotide (siRNA) solution was prepared at 1.31 mg/ml in 25 mMacetate buffer at pH 4.0. The lipid mixture was prepared to contain 90mol % cationic lipid (DLin-KC2-DMA) and 10 mol % PEG-c-DMA (10 mM totallipid dissolved in ethanol). The two solutions were mixed using themicrofluidic mixer at a total flow rate 2 ml/min and diluted 2-fold with25 mM acetate buffer, pH 4.0, to bring ethanol down to about 23 vol %,forming the initial or core nanoparticle. Sequential assembly wasperformed by taking this initial lipid particle suspension and mixing itwith another lipid solution containing an anionic lipiddioleoylphosphatidylserine (DOPS) dissolved in methanol and furtherdiluting to approximately 25 vol % solvent (methanol and ethanol). Thesecond lipid, DOPS, was added at about 4× molar excess to the cationiclipid. The sequential assembly process was repeated by alternatingbetween the cationic lipid and anionic lipid.

Particle size was determined by dynamic light scattering using a MalvernZetasizer Nano-ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK). Number-weighted distribution data was used. Zeta potential whichprovides a measure of the surface charge of the LNP systems was measuredwith the Malvern Zetasizer using disposable capillary cells (DTS1060,Malvern Instruments Ltd.). The LNP systems were diluted to approximately0.3 mg/ml total lipid in 25 mM acetate buffer, pH 4.0.

Example 5

Preparation and Characteristics of a Representative Lipid Particle

In this example, a representative lipid particle the inventionconsisting only of a cationic lipid and a nucleic acid (DLin-KC2-DMA —siRNA), are described.

The siRNA solution was prepared at 0.38 mg/ml in 25 mM acetate buffer,pH 4.0. The lipid solution was prepared to contain DLin-KC2-DMA at aconcentration of 10 mM in ethanol. The siRNA-to-lipid ratio was 0.06(wt/wt). Each solution was input into the microfluidic mixer at equalflow rates and a total flow rate of 2 ml/min. The sample was furtherdiluted with 25 mM acetate buffer, pH 4.0, to bring ethanol content to25 vol %.

Particle size was determined by dynamic light scattering using a Nicompmodel 370 Submicron Particle Sizer (Particle Sizing Systems, SantaBarbara, Calif., USA). Sample measurement was performed in 25 mM acetateand number-weighted distribution data was used. The particles had a meanparticle diameter of 14.2 nm, a coefficient of variance of 0.487, andχ²of 1.93.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method for introducing a polynucleic acid into a cell, comprisingcontacting a cell with a lipid particle, comprising an electron densecore comprising a polynucleic acid and an ionizable lipid, the corecoated with one or more polar lipids comprising a PEG-lipid.
 2. Themethod of claim 1, wherein the ionizable lipid is an amino lipid.
 3. Themethod of claim 1, wherein the ionizable lipid is a dilinoleyl aminolipid.
 4. The method of claim 1, wherein the ionizable lipid is selectedfrom the group consisting of DODAC, DOTMA, DDAB, DOTAP, DOTAP.Cl,DC-Chol, DOSPA, DOGS, DOPE, DODAP, DODMA, and DMRIE.
 5. The method ofclaim 1, wherein the ionizable lipid has the formula:

wherein R₁ and R₂ are either the same or different and independentlyoptionally substituted C₁₀-C₂₄ alkyl, optionally substituted C₁₀-C₂₄alkenyl, optionally substituted C₁₀-C₂₄ alkynyl, or optionallysubstituted C₁₀-C₂₄ acyl; R₃ and R₄ are either the same or different andindependently optionally substituted C₁-C₆ alkyl, optionally substitutedC₂-C₆ alkenyl, or optionally substituted C₂-C₆ alkynyl or R₃ and R₄ mayjoin to form an optionally substituted heterocyclic ring of 4 to 6carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R₅is either absent or present and when present is hydrogen or C₁-C₆ alkyl;m, n, and p are either the same or different and independently either 0or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0,1 , 2, 3, or 4; and Y and Z are either the same or different andindependently O, S, or NH.
 6. The method of claim 1, wherein thePEG-lipid is selected from the group consisting of PEG-modifiedphosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modifiedceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, andPEG-modified dialkylglycerols.
 7. The method of claim 1, wherein thePEG-lipid is selected from the group consisting of PEG-c-DMA,PEG-c-DOMG, and PEG-s-DMG.
 8. The method of claim 1, comprising fromabout 1 to about 5 mole percent PEG-lipid.
 9. The method of claim 1,comprising about 1 mole percent PEG-lipid.
 10. The method of claim 1,wherein the polar lipids are selected from the group consisting ofneutral lipids and sterols.
 11. The method of claim 1, wherein the polarlipids comprise a neutral lipid selected from the group consisting ofdiacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides. 12.The method of claim 1, wherein the polar lipids comprise a neutral lipidselected from DSPC and DOPC.
 13. The method claim 1, wherein the polarlipids comprise a sterol.
 14. The method of claim 1, wherein thepolynucleic acid is a DNA, an RNA, a locked nucleic acid, a nucleic acidanalog, or a plasmid capable of expressing a DNA or an RNA.
 15. Themethod of claim 1, wherein the polynucleic acid is ssDNA or dsDNA. 16.The method of claim 1, wherein the polynucleic acid is siRNA ormicroRNA.
 17. The method particle of claim 1, wherein the polynucleicacid is an oligonucleotide.
 18. The method of claim 1, wherein thepolynucleic acid is an antisense oligonucleotide.
 19. The method ofclaim 1, wherein the particle has a diameter from about 30 nm to about200 nm.
 20. The method of claim 1, wherein the particle has a diameterof about 80 nm.